Diazonamide analogs

ABSTRACT

Diazonamide analogs having anti-mitotic activity, useful for the treatment of cancer and other proliferative disorders, and related pharmaceutical compositions are provided.

RELATED APPLICATION

This application claims priority to U.S. 61/478,059, filed Apr. 22,2011, and having the same title and inventors.

INTRODUCTION

Diazonamide A is a mitotic spindle-disrupting agent first isolated fromthe marine organism Diazona angulata, having the structure:

The preparation of diazonamide analogs via macrocyclic indolineintermediates bearing a carbobenzyloxy (Cbz) or o-nitrophenylsulfonylprotected amino group has been previously described. U.S. Pat. No.7,022,720 and U.S. Pat. No. 7,517,895 correctly disclose the structureof diazonamide A and describe the synthesis of some of its analogs. U.S.Pat. No. 7,851,620 (continued with U.S. Ser No. 12/896,898) describessynthetic methods for the preparation of diazonamide analogs viaindoline intermediates. U.S. Pat. No. 7,538,129 describes diazonamide Aanalogs. U.S. Ser No. 12/432,615 is a related pending applicationdisclosing indoline, which lack the rigid macrocyclic structure bridgingthe A- and E-rings of the diazonamide skeleton. Disclosed here arecompounds of formula (I) and additional novel diazonamide analogs whichpossess potent cytotoxic activity and are useful for the treatment ofcell proliferative disorders.

SUMMARY OF THE INVENTION

The present invention is directed towards compounds of formula (I) andpharmaceutically acceptable salts and conjugates thereof, pharmaceuticalcompositions comprising a compound of formula (I) and/or a salt orconjugate thereof, modified forms of such compounds conjugated tostabilizing or targeting agents, and methods of making and using thesecompounds and formulations, wherein formula (I) is:

or a pharmaceutically acceptable salt or conjugate thereof;wherein:R¹ is optionally substituted C1-C4 alkyl;R² is H, or optionally substituted C1-C4 alkyl;R³ is C1-C12 alkyl, C1-C12 heteroalkyl, C2-C12 alkenyl, C2-C12heteroalkenyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, C4-C12cycloalkylalkyl, C4-C12 heterocyclylalkyl, C6-C12 aryl, C5-C12heteroaryl, C7-C14 arylalkyl, or C6-C14 heteroarylalkyl, each of whichmay be optionally substituted;R⁴ is H, or optionally substituted C1-C4 alkyl;R⁵ is optionally substituted C6-C12 aryl or optionally substitutedC5-C12 heteroaryl;R⁶ is H, or optionally substituted C1-C4 alkyl;each Y and Y′ is independently halo, OH, C1-C4 alkoxy, or C1-C8 alkyl,C2-C8 alkenyl, C2-C8 alkynyl, C6-C12 aryl, or C7-C14 arylalkyl, or aheteroform of one of these, each of which may be optionally substituted;m is 0-4; andm′ is 0-3.

The invention encompasses all combinations of various preferredembodiments/substitutions of formula (I) described herein.

In a further aspect, the invention provides a pharmaceutical compositioncomprising at least one compound of formula (I) or a disclosedembodiment thereof, and a pharmaceutically acceptable excipient.

In some embodiments, the compound of formula (I) or a disclosedembodiment thereof is a compound in one of the Tables provided herein,or a pharmaceutically acceptable salt or conjugate of one of thesecompounds.

In another aspect, the invention provides a method for treating orameliorating a cell proliferative disorder, comprising administering toa subject in need thereof a therapeutically effective amount of at leastone compound of formula (I) or a disclosed embodiment thereof or a salt,conjugate, or pharmaceutical composition thereof. In some embodiments,the amount administered is sufficient to inhibit cell proliferation. Inother embodiments, the amount is sufficient to slow tumor growth orreduce tumor size. In some embodiments, the compound of formula (I) or adisclosed embodiment thereof is used in combination with anotherchemotherapeutic agent or approach.

Provided also are methods for inhibiting cell proliferation in a cell,comprising contacting the cell with a compound of one of the formuladescribed herein, or a salt, or conjugate thereof, in an amounteffective to inhibit cell proliferation. In some embodiments, the cellsare in a cell line, such as a cancer cell line (e.g., a cell linederived from breast, prostate, pancreatic, lung, or hematopoieticcancers, etc.). In some embodiments, the cells are in a tissue, an insome such embodiments, the tissue can be in a subject. In otherembodiments, the cells are in a tumor, and sometimes are in a tumor in asubject.

Provided also are methods for treating cancer in a subject in need ofsuch treatment, comprising: administering to the subject atherapeutically effective amount of a compound of formula (I) or adisclosed embodiment thereof or a salt or conjugate thereof, asdescribed herein, in an amount that is effective to treat or amelioratesaid cancer.

The invention further provides methods for treating or ameliorating acondition related to aberrant cell proliferation. For example, providedare methods of treating or ameliorating a cell proliferative disorder ina subject, comprising administering a compound of formula (I) or adisclosed embodiment thereof or a salt or conjugate thereof, asdescribed herein, to a subject in need thereof in an amount effective totreat or ameliorate the condition.

In the methods described herein, the subject may be a research animal(e.g., rodent, dog, cat, monkey), optionally containing a tumor such asa xenograft tumor (e.g., human tumor), for example, or may be a human.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows data for subject compounds in an HCC461 human lungcarcinoma xenograft model in mice.

FIG. 2 shows data for subject compounds in a Miapaca pancreatic cancerxenograft model in mice.

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. It is to be understood thatthe terminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting. It is further to beunderstood that unless specifically defined herein, the terminology usedherein is to be given its traditional meaning as known in the relevantart.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless indicated otherwise.

As used herein, the term “subject” refers to a human or animal subject.In preferred embodiments, the subject is human.

The terms “treat”, “treating” or “treatment” in reference to aparticular disease or disorder include prevention of the disease ordisorder, and/or lessening, improving, ameliorating, alleviating orremoving the symptoms and/or pathology of the disease or disorder.

The term “therapeutically effective amount” or “effective amount” isintended to mean that amount of a drug or pharmaceutical agent that willelicit a biological or medical response of a cell, tissue, system,animal or human that is being sought by a researcher, veterinarian,medical doctor or other clinician. The terms also can refer to reducingor stopping a cell proliferation rate (e.g., slowing or halting tumorgrowth) or reducing the number of proliferating cancer cells (e.g.,removing part or all of a tumor). Sometimes, the rate or cellproliferation is reduced by 10%, 20%, 30%, 40%, 50%, 60%, or 70% ormore. Sometimes, the number of proliferating cells is reduced by 10%,20%, 30%, 40%, 50%, 60%, or 70% or more.

As used herein, the terms “alkyl,” “alkenyl” and “alkynyl” includestraight-chain, branched-chain and cyclic monovalent hydrocarbylradicals, and combinations of these, which contain only C and H whenthey are unsubstituted. Examples include methyl, ethyl, isopropyl,isobutyl, tert-butyl, cyclohexyl, cyclopentylethyl, 2-propenyl,3-butynyl, and the like. The total number of carbon atoms in each suchgroup is sometimes described herein, e.g., when the group can contain upto twelve carbon atoms it may be described as 1-12C or as C1-C12 or asC1-12 or as C₁₋₁₂. When heteroatoms (typically N, O and S) are allowedto replace carbon atoms of an alkyl, alkenyl or alkynyl group, as inheteroalkyl groups, for example, the numbers describing the group,though still written as e.g. C1-C6, represent the sum of the number ofcarbon atoms in the group plus the number of such heteroatoms that areincluded as replacements for carbon atoms in the ring or chain beingdescribed.

Typically, the alkyl, alkenyl and alkynyl substituents of the inventioncontain 1-12C (alkyl) or 2-12C (alkenyl or alkynyl). Preferably theycontain 1-8C (alkyl) or 2-8C (alkenyl or alkynyl). Sometimes theycontain 1-4C (alkyl) or 2-4C (alkenyl or alkynyl). A single group caninclude more than one type of multiple bond, or more than one multiplebond; such groups are included within the definition of the term“alkenyl” when they contain at least one carbon-carbon double bond, andthey are included within the term “alkynyl” when they contain at leastone carbon-carbon triple bond.

“Heteroalkyl”, “heteroalkenyl”, and “heteroalkynyl” and the like aredefined similarly to the corresponding hydrocarbyl (alkyl, alkenyl andalkynyl) groups, but the ‘hetero’ terms refer to groups that contain oneor more heteroatoms selected from O, S and N and combinations thereof,within the backbone residue; thus at least one carbon atom of acorresponding alkyl, alkenyl, or alkynyl group is replaced by one of thespecified heteroatoms to form a heteroalkyl, heteroalkenyl, orheteroalkynyl group. Preferably, each heteroalkyl, heteroalkenyl andheteroalkynyl group contains only 1-2 heteroatoms as part of theskeleton of backbone of the heteroalkyl group, i.e., not includingsubstituents that may be present. Hence, heteroalkyls include alkoxylssuch as O-alkyl, alkyl ethers, secondary and tertiary alkyl amines,alkyl sulfides, alkyl sulfonyls, and the like.

The typical and preferred sizes for heteroforms of alkyl, alkenyl andalkynyl groups are generally the same as for the correspondinghydrocarbyl groups, and the substituents that may be present on theheteroforms are the same as those described above for the hydrocarbylgroups. Where such groups contain N, the nitrogen atom may be present asNH or it may be substituted if the heteroalkyl or similar group isdescribed as optionally substituted. Where such groups contain S, thesulfur atom may optionally be oxidized to SO or SO₂ unless otherwiseindicated. For reasons of chemical stability, it is also understoodthat, unless otherwise specified, such groups do not include more thanthree contiguous heteroatoms as part of the heteroalkyl chain, althoughan oxo group may be present on N or S as in a nitro or sulfonyl group.Thus —C(O)NH₂ can be a C2 heteroalkyl group substituted with ═O; and—SO₂NH— can be a C2 heteroalkylene, where S replaces one carbon, Nreplaces one carbon, and S is substituted with two ═O groups.

While “alkyl” as used herein includes cycloalkyl and cycloalkylalkylgroups, the term “cycloalkyl” may be used herein to specificallydescribe a saturated or partially saturated, monocyclic or fused orspiro polycyclic, carbocycle that is connected via a ring carbon atom,and “cycloalkylalkyl” may be used to describe a carbocyclic non-aromaticgroup that is connected to the base molecule through an alkyl linker.Similarly, “heterocyclyl” may be used to describe a non-aromatic cyclicgroup that contains at least one heteroatom as a ring member and that isconnected to the molecule via a ring atom of the cyclic group, which maybe C or N; and “heterocyclylalkyl” may be used to describe such a groupthat is connected to another molecule through an alkyl linker. The sizesand substituents that are suitable for the cycloalkyl, cycloalkylalkyl,heterocyclyl, and heterocyclylalkyl groups are the same as thosedescribed above for alkyl groups. Frequently, cycloalkyl andheterocyclyl groups are C3-C8, and cycloalkylalkyl or heterocyclylalkylgroups are C4-C12. The size of a cycloalkylalkyl or heterocyclylalkylgroup describes the total number of carbon atoms or of carbon atoms plusheteroatoms that replace carbon atoms of an alkyl, alkenyl, alkynyl,cycloalkyl, or cycloalkylalkyl portion. As used herein, these terms alsoinclude rings that contain a double bond or two, as long as the ring isnot aromatic.

As used herein, “acyl” encompasses groups comprising an alkyl, alkenyl,alkynyl, aryl or arylalkyl radical attached at one of the two availablevalence positions of a carbonyl carbon atom (which may be depictedherein as —C(═O)R, —C(O)R, or COR) where R is an alkyl, alkenyl,alkynyl, aryl, or arylalkyl group, and heteroacyl refers to thecorresponding groups wherein at least one carbon other than the carbonylcarbon has been replaced by a heteroatom chosen from N, O and S. Thusheteroacyl includes, for example, —C(═O)OR and —C(═O)NR₂ as well as—C(═O)-heteroaryl. Also included within the definition of heteroacylgroups are thioacyl substituents, e.g., —C(═S)R, and imine groups, e.g.,—C(═NH)R.

Acyl and heteroacyl groups are bonded to any group or molecule to whichthey are attached through the open valence of the carbonyl carbon atom.Typically, they are C1-C8 acyl groups, which include formyl, acetyl,trifluoroacetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl groups,which include methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. Thehydrocarbyl groups, aryl groups, and heteroforms of such groups thatcomprise an acyl or heteroacyl group can be substituted with thesubstituents described herein as generally suitable substituents foreach of the corresponding component of the acyl or heteroacyl group.

“Aromatic” moiety or “aryl” moiety refers to a monocyclic or fusedbicyclic moiety having the well-known characteristics of aromaticity;examples include phenyl and naphthyl. Carbocyclic aryl rings and ringsystems typically 6-12 carbon ring atoms, and may include a saturated orpartially unsaturated carbocyclic ring fused to an aromatic ring, e.g.,a tetrahydronaphthalene, indane or indene ring system. Similarly,“heteroaromatic” and “heteroaryl” refer to such monocyclic or fusedbicyclic ring systems which contain as ring members one or moreheteroatoms selected from O, S and N. The inclusion of a heteroatompermits aromaticity in 5-membered rings as well as 6-membered rings.Typical heteroaromatic systems include monocyclic C5-C6 aromatic groupssuch as pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazinyl, thienyl,furanyl, pyrrolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl,isoxazolyl, imidazolyl, triazolyl, thiadiazolyl, oxadiazolyl, andtetrazolyl rings, and the fused bicyclic moieties formed by fusing oneof these monocyclic groups with a phenyl ring or with any of theheteroaromatic monocyclic groups to form a C8-C10 bicyclic group such asindolyl, benzimidazolyl, indazolyl, benzotriazolyl, isoquinolinyl,quinolinyl, benzothiazolyl, benzofuranyl, benzothienyl, benzisoxazolyl,pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like.Any monocyclic or fused ring bicyclic system which has thecharacteristics of aromaticity in terms of electron distributionthroughout the ring system is included in this definition. It alsoincludes bicyclic groups where at least one ring has the characteristicsof aromaticity, even though it may be fused to a nonaromatic ring.Typically, the ring systems contain 5-12 ring member atoms. Preferablythe monocyclic aryl and heteroaryl groups contain 5-6 ring members, andthe bicyclic aryl and heteroaryl groups contain 8-10 ring members.

Similarly, “arylalkyl” and “heteroarylalkyl” refer to aromatic andheteroaromatic ring systems which are bonded to their attachment pointthrough a linking group such as an alkylene, including substituted orunsubstituted, saturated or unsaturated, cyclic or acyclic linkers.Typically the linker is C1-C8 alkyl or a heteroform thereof, preferablya C1-C4 alkyl. These linkers may also include a carbonyl group, thusmaking them able to provide substituents as an acyl or heteroacylmoieties.

“Arylalkyl” groups as used herein are hydrocarbyl groups if they areunsubstituted, and are described by the total number of carbon atoms inthe ring and alkylene or similar linker. Thus a benzyl group is aC7-arylalkyl group, and phenylethyl is a C8-arylalkyl. Preferably, anarylalkyl group includes one or two optionally substituted phenyl ringsand a C1-C4 alkylene that is unsubstituted or is substituted with one ortwo C1-C4 alkyl groups or C1-C4 heteroalkyl groups, where the alkyl orheteroalkyl groups can optionally cyclize to form a ring such ascyclopropane, dioxolane, or oxacyclopentane, and wherein the alkyl orheteroalkyl groups may be optionally fluorinated. Examples of arylalkylgroups include optionally substituted benzyl, phenylethyl,diphenylmethyl, and triphenylmethyl groups. Optional substituents whenpresent on the aryl ring of an arylalkyl group are the same as thosedescribed herein for an aryl ring. Arylalkyl groups typically containfrom 7-20 atoms, preferably 7-14 atoms.

“Heteroarylalkyl” as described above refers to a moiety comprising anaryl group that is attached through a linking group, and differs from“arylalkyl” in that at least one ring atom of the aryl moiety or oneatom in the linking group is a heteroatom selected from N, O and S. Theheteroarylalkyl groups are described herein according to the totalnumber of atoms in the ring and linker combined, and they include arylgroups linked through a heteroalkyl linker; heteroaryl groups linkedthrough a hydrocarbyl linker such as an alkylene; and heteroaryl groupslinked through a heteroalkyl linker. For example, heteroaryl groupsinclude pyridylmethyl, pyridylethyl, —O-benzyl, and the like.Heteroarylalkyl groups typically contain from 6-20 atoms, preferably6-14 atoms.

“Alkylene” as used herein refers to a divalent hydrocarbyl group;because it is divalent, it can link two other groups together. Typicallyit refers to —(CH₂)_(n)— where n is 1-8 and preferably n is 1-4, thoughwhere specified, an alkylene can also be substituted by other groups,and can be of other lengths, and the open valences need not be atopposite ends of a chain. Thus —CH(Me)- and —C(Me)₂- may also bereferred to as alkylenes, as can a cyclic group such ascyclopropan-1,1-diyl. However, for clarity, a three-atom linker that isan alkylene group, for example, refers to a divalent group in which theavailable valences for attachment to other groups are separated by threeatoms such as —(CH₂)₃—, i.e., the specified length represents the numberof atoms linking the attachment points rather than the total number ofatoms in the hydrocarbyl group: —C(Me)₂- would thus be a one-atomlinker, since the available valences are separated by only one atom.Where an alkylene group is substituted, the substituents include thosetypically present on alkyl groups as described herein, thus —C(═O)— isan example of a one-carbon substituted alkylene. Where it is describedas unsaturated, the alkylene may contain one or more double or triplebonds.

“Heteroalkylene” as used herein is defined similarly to thecorresponding alkylene groups, but the ‘hetero’ terms refer to groupsthat contain one or more heteroatoms selected from O, S and N andcombinations thereof, within the backbone residue; thus at least onecarbon atom of a corresponding alkylene group is replaced by one of thespecified heteroatoms to form a heteroalkylene group. Thus, —C(═O)NH— isan example of a two-carbon substituted heteroalkylene, where N replacesone carbon, and C is substituted with a ═O group.

“Heteroform” as used herein refers to a derivative of a group such as analkyl, aryl, or acyl, wherein at least one carbon atom of the designatedcarbocyclic group has been replaced by a heteroatom selected from N, Oand S. Thus the heteroforms of alkyl, alkenyl, cycloalkyl, alkynyl,acyl, aryl, and arylalkyl are heteroalkyl, heteroalkenyl, heterocyclyl,heteroalkynyl, heteroacyl, heteroaryl, and heteroarylalkyl,respectively. It will be understood that the heteroform of an aryl orarylalkyl moiety may contain one less “C” atom than the correspondingall carbon system, because the inclusion of a heteroatom permitsaromaticity in 5-membered rings. For example, the heteroform of C6-C12aryl is C5-C12 heteroaryl, and the heteroform of C7-C20 arylalkyl isC6-C20 heteroarylalkyl. It is understood that no more than two N, O or Satoms are ordinarily connected sequentially, except where an oxo groupis attached to N or S to form a nitro or sulfonyl group, or in the caseof certain heteroaromatic rings, such as triazine, triazole, tetrazole,oxadiazole, thiadiazole, and the like.

Unless otherwise indicated, the term “oxo” refers to ═O.

“Halo”, as used herein, includes fluoro, chloro, bromo and iodo. Fluoro,chloro, and bromo are often preferred.

“Amino” as used herein refers to NH₂, but where an amino is described as“substituted” or “optionally substituted”, the term includes NR₂ whereineach R is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl,or arylalkyl group or a heteroform of one of these groups, as furtherdefined herein, each of which may be optionally substituted with thesubstituents described herein as suitable for the corresponding type ofgroup. The term also includes forms wherein the two R groups on onenitrogen atom (i.e., NR₂) are linked together to form a 3-8 memberedmonocyclic azacyclic ring or an 8-12 membered bicyclic fused azacyclicring system, each of which may be saturated, unsaturated or aromatic andwhich may contain 1-3 heteroatoms including the azacylic ring nitrogenatom independently selected from N, O and S as ring members (i.e., 0-2heteroatoms selected from N, O and S in addition to the nitrogen atom ofthe azacyclic ring), and which may be optionally substituted with thesubstituents described as suitable for alkyl groups or, if NR₂ comprisesan aromatic group, it may be optionally substituted with thesubstituents described as typical for aryl or heteroaryl groups.Preferred such azacyclic rings include pyrrolidine, piperidine,homopiperidine, morpholine, thiomorpholine, piperazine, andhomopiperazine.

Amino groups may optionally be in a protected or unprotected form. Oneof skill in the art would appreciate that appropriate amine protectinggroups may vary depending on the functionality present in the particularmolecule and the nature of the amino group. Suitably protected aminesmay include, for example, amines protected as carbamates (e.g.,tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),fluorenylmethyloxy-carbonyl (Fmoc), allyloxycarbonyl (Alloc) or(trialkylsilyl)ethoxycarbonyl), carboxamides (e.g., formyl, acyl ortrifluoroacetyl, benzoyl), sulfonamides, phthalimides, succinimides,Schiff's base derivatives, and the like. Also included are alkyl orallyl amines, as well as trialkylsilyl protected amines.

Where an amine is present in protected form, it is sometimes desirableto remove the protecting group. Thus, the methods of the presentinvention also optionally include a step of removing any protectinggroups on an amine or aminoalkyl group.

The terms “alkylsulfonyl” and “arylsulfonyl” as used herein refer tomoieties of the form —SO₂alkyl or —SO₂aryl, where alkyl and aryl aredefined as above. Optionally fluorinated C₁₋₄alkyl, and optionallysubstituted phenyl groups are preferred for sulfonyl moieties. Thephenyl groups of an arylsulfonyl moiety may be optionally substitutedwith one or more substituents suitable for an aryl ring; for example,they may be substituted by halo, methyl, nitro, alkoxy, amino, or thelike. Such sulfonyl moieties, when present on oxygen form sulfonates.Such sulfonyl moieties form sulfonamides when present on nitrogen, andsulfones when present on carbon. Representative sulfonates include,e.g., —OSO₂Me (mesylate), —OSO₂CF₃ (triflate), —OSO₂tolyl (tosylate),and the like.

The term “alkoxycarbonyl” as used herein refers to a moiety of the form—COOR′, where R′ is C1-C8 alkyl, C2-C8 alkenyl, C5-C6 aryl, or C7-C14arylalkyl, trialkylsilyl, or the like, each of which may be optionallysubstituted. When present on nitrogen, such alkoxycarbonyl moieties formcarbamates, which are frequently used as nitrogen protecting groups. Insome such embodiments, R′ may be optionally halogenated C1-C4 alkyl(e.g., tert-butyl, methyl, ethyl, 2,2,2-trichloroethyl,1,1-dimethyl-2,2,2-trichloroethyl), allyl, optionally substitutedbenzyl, fluorenylmethyl, or trialkylsilyl (e.g., triisopropylsilyl,triethylsilyl, tert-butyldimethylsilyl). When present on carbon, suchmoieties may also be referred to as carboxylate esters, carboalkoxygroups, or the like. In some embodiments containing a carboxylate esterfunctional group, R′ is preferably a C₁₋₄ alkyl group. In some suchembodiments, R′ is methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, s-butyl or t-butyl.

The term “substituted” means that the specified group or moiety bearsone or more non-hydrogen substituents. The term “unsubstituted” meansthat the specified group bears no such substituents.

“Optionally substituted” as used herein indicates that the particulargroup or groups being described may have no non-hydrogen substituents,or the group or groups may have one or more non-hydrogen substituents(i.e., the group may be substituted or unsubstituted). If not otherwisespecified, the total number of such substituents that may be present isequal to the number of H atoms present on the unsubstituted form of thegroup being described. Where an optional substituent is attached via adouble bond, such as a carbonyl oxygen (═O), the group takes up twoavailable valences, so the total number of substituents that may beincluded is reduced according to the number of available valences.

Alkyl, alkenyl and alkynyl groups are often substituted to the extentthat such substitution makes sense chemically. Typical substituentsinclude, but are not limited to, halo, OH, ═O, ═N—CN, ═N—OR, ═NR, OR,NR₂, SR, SOR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂, NRCOOR, NRCOR, CN, COOR,CONR₂, OOCR, COR, and NO₂, wherein each R is independently H, optionallyfluorinated C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8heteroalkynyl, C6-C12 aryl, C5-C12 heteroaryl, C5-C20 arylalkyl, orC5-C20 heteroarylalkyl, and each R is optionally substituted with one ormore groups selected from halo, OH, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′₂,SR′, SOR′, SO₂R′, SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN,COOR′, CONR′₂, OOCR′, COR′, and NO₂, wherein each R′ is independently H,optionally fluorinated C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8heteroacyl, C6-C12 aryl, C5-C12 heteroaryl, C5-C20 arylalkyl, or C5-C20heteroarylalkyl. Alkyl, alkenyl and alkynyl groups can also besubstituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C12 aryl or C5-C12heteroaryl, each of which can be substituted by the substituents thatare appropriate for the particular group.

Preferred substituents when present on an alkyl, alkenyl or alkynylgroup, or a heteroform of one of these, include halo, OH, ═O, OR, SR,and NR₂, where R is defined as above; sometimes, R is H, optionallyfluorinated C1-C4 alkyl, or optionally fluorinated C1-C4 acyl.Particularly preferred substituents when present on R³ include OH, ═O,C1-C4 alkoxy, OAc, NHAc, NH₂, and NHMe. Sometimes, optional substituentspresent on an alkyl, alkenyl or alkynyl group, or a heteroform of one ofthese, include NRSO₂R, NRCONR₂, COOR, or CONR₂, where R is defined asabove; preferably, each R is independently H, optionally fluorinatedC1-C4 alkyl, or is C6-C12 aryl, C5-C12 heteroaryl, C7-C20 arylalkyl, orC6-C20 heteroarylalkyl, each of which may be optionally substituted.

Aryl, heteroaryl and heterocyclyl moieties may be substituted with avariety of substituents including optionally fluorinated C1-C8 alkyl,C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 acyl, and heteroforms of these,C6-C12 aryl, C5-C12 for heteroaryl, C6-20 arylalkyl (C5-20 forheteroarylalkyl), each of which can itself be further substituted; othersubstituents for aryl and heteroaryl moieties include halo, OH, OR,CH₂OH, CH₂OR, CH₂NR₂, NR₂, SR, SOR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂,NRCOOR, NRCOR, CN, COOR, CONR₂, OOCR, C(O)R, and NO₂, wherein each R isindependently H, optionally fluorinated C1-C8 alkyl, C2-C8 heteroalkyl,C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl,C6-C12 aryl, C5-C12 heteroaryl, C7-C20 arylalkyl, or C6-C20heteroarylalkyl, and each R is optionally substituted as described abovefor alkyl groups. The substituent groups on an aryl or heteroaryl groupmay of course be further substituted with the groups described herein assuitable for each type of group that comprises the substituent.Preferred substituents when present on an aryl, heteroaryl andheterocyclyl moieties include halo, OH, OR, CH₂OH, CH₂OR, CH₂NR₂, SR,NR₂, CN, COOR, CONR₂, and NO₂, where R is defined as above, oroptionally substituted C6-C12 aryl or C5-C12 heteroaryl ring.

Where an arylalkyl or heteroarylalkyl group is described as optionallysubstituted, the substituents may be on either the alkyl or heteroalkylportion or on the aryl or heteroaryl portion of the group. Thesubstituents optionally present on the alkyl or heteroalkyl portion arethe same as those described above for alkyl groups generally; thesubstituents optionally present on the aryl or heteroaryl portion arethe same as those described above for aryl groups generally.

The invention encompasses isomers of the subject compounds, particularlystereoisomers, such as wherein the carbon atom bearing the substituentR¹ in formula (I) or the corresponding atom in disclosed embodiments offormula (I), has the (S)-configuration.

The present invention provides novel indoline analogs of formula (I),which are useful for the treatment or amelioration of proliferativedisorders, in particular, cancer.

The invention encompasses all combinations of preferred embodiments andpreferred substituents described herein.

Preferably, R¹ is optionally substituted C2-C4 alkyl, preferably C2-C4alkyl, preferably propyl or butyl, preferably isopropyl or t-butyl.

Preferably, R², R⁴ and R⁶ are independently H or methyl, preferably H. Asubstituent at R⁴ may function as a protecting group, and methodsdescribed herein include an optional deprotection step to remove anyprotecting groups present on the molecule.

Preferably, R³ is a substituted methyl of the general formula(—CR^(a)R^(b)R^(c)) wherein R^(a) is OH, OR, CH₂OR, SR, and NR₂, whereeach R is independently H, optionally halogenated (preferablyfluorinated or chlorinated) C1-C4 alkyl, or optionally halogenated C1-C4acyl, and preferably OH; and each of R^(b) and R^(c) is independently H,C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8cycloalkylalkyl, C6-C12 aryl, C7-C14 arylalkyl, or a heteroform of oneof these, each of which may be optionally substituted, and preferably Hor C1-C4 lower alkyl, more preferably H and isopropyl or t-butyl,respectively; or R^(b) and R^(c) may be taken together with the carbonto which they are attached to form a C3-C8 cycloalkyl or a C3-C8heterocyclyl ring, which may be optionally substituted. For example,R^(b) and R^(c) may be taken together to form an optionally substitutedcyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, tetrahydrofuran, tetrahydropyran, tetrahydrothiofuran,tetrahydrothiopyran, pyrrolidine, or piperidine ring, and the like. In apreferred embodiment, each of R^(b) and R^(c) are taken together to forma cyclohexyl or a cyclopentyl ring. In some embodiments, the ring formedby R^(b) and R^(c) may be fused to a substituted or unsubstituted phenylring to provide, for example, and indenyl or tetrahydronaphthyl ringsystem.

In other preferred embodiments, R³ is C1-C4 alkyl, C3-C6 cycloalkyl,C4-C8 cycloalkylalkyl, or C6-C8 arylalkyl, each of which may beoptionally substituted. In preferred embodiments, the alkyl groupcomprising part of R³ is substituted with at least one substituentselected from the group consisting of OH, OR, CH₂OR, SR, and NR₂, whereeach R is independently H, optionally fluorinated C1-C4 alkyl, oroptionally fluorinated C1-C4 acyl. Preferably R³ is substituted with atleast one substituent selected from the group consisting of OH, OMe,OAc, NH₂, NHMe, CH₂OH and NHAc. In more specific embodiments, R³ is aC1-C8, preferably C1-C4, more preferably C2-C3, most preferably C2straight chain, branched, or cycloalkyl group, each of which issubstituted on the carbon atom adjacent to the carbonyl group that ispart of R⁵ with OH, OMe, OAc, NH₂, NHMe, CH₂OH or NHAc, preferably OH.

Preferably R⁵ is an optionally substituted phenyl, naphthyl,benzimidazole, benzoxazole, benzthiazole, pyridinyl, pyrimidinyl,pyrazinyl or pyridazinyl ring, and more preferably, R⁵ is an optionallysubstituted oxazole, oxazoline, thiazole, thiazoline, pyrazole,pyrazoline, imidazole, imidazoline, pyrrole, pyrroline, isoxazole,isoxazoline, isothiazole, isothiazoline, oxadiazole, thiadiazole,triazole or tetrazole ring.

Preferred substituents include halo, nitro, cyano, or optionallyfluorinated C1-C4 alkyl, optionally fluorinated C1-C4 alkoxy, COOR^(S),CONR⁹ ₂, C6-C12 aryl or C5-C12 heteroaryl, each of which may beoptionally substituted; where R⁸ is H, or C1-C8 alkyl, C2-C8 alkenyl,C6-C12 aryl, or C7-C14 arylalkyl, or a heteroform of one of these, eachof which may be optionally substituted; and each R⁹ is independently H,or C1-C12 alkyl, C1-C12 heteroalkyl, C2-C12 alkenyl, C2-C12heteroalkenyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, C4-C12cycloalkylalkyl, C4-C12 heterocyclylalkyl, C6-C12 aryl, C5-C12heteroaryl, C7-C14 arylalkyl, or C6-C14 heteroarylalkyl, each of whichmay be optionally substituted; or two R⁹ on the same N can cyclize toform an optionally substituted 3- to 8-membered azacyclic ring,optionally containing an additional heteroatom selected from N, O, and Sas a ring member; preferred such azacyclic rings include pyrrolidine,piperidine, homopiperidine, morpholine, thiomorpholine, piperazine, andhomopiperazine.

In certain preferred embodiments, R⁵ is an optionally substitutedoxazole or thiazole ring. In some such embodiments, R⁵ is an oxazolering substituted with an optionally substituted C6-C12 aryl or C5-C12heteroaryl ring. In some embodiments, R⁵ is an oxazole ring substitutedwith one or more alkyl, halo, carboxylic acid, ester or amidesubstituents.

In specific embodiments of formula (I), R⁵ is an optionally substitutedheterocyclic or heteroaromatic ring of the formula:

wherein Q is O, S or NR¹³, where R¹³ is H or C1-C4 alkyl; each R¹⁴ isindependently halo, nitro, cyano, or optionally fluorinated C1-C4 alkyl,optionally fluorinated C1-C4 alkoxy, COOR⁸, CONR⁹ ₂, C6-C12 aryl orC5-C12 heteroaryl, each of which may be optionally substituted; where R⁸is H, or C1-C8 alkyl, C2-C8 alkenyl, C6-C12 aryl, or C7-C14 arylalkyl,or a heteroform of one of these, each of which may be optionallysubstituted; and each R⁹ is independently H, or C1-C12 alkyl, C1-C12heteroalkyl, C2-C12 alkenyl, C2-C12 heteroalkenyl, C3-C8 cycloalkyl,C3-C8 heterocyclyl, C4-C12 cycloalkylalkyl, C4-C12 heterocyclylalkyl,C6-C12 aryl, C5-C12 heteroaryl, C7-C14 arylalkyl, or C6-C14heteroarylalkyl, each of which may be optionally substituted; or two R⁹on the same N can cyclize to form an optionally substituted 3- to8-membered azacyclic ring, optionally containing an additionalheteroatom selected from N, O, and S as a ring member; p is 0-3; and qis 0 to 4. Such azacyclic rings may be saturated, unsaturated oraromatic; preferred such azacyclic rings include pyrrolidine,piperidine, homopiperidine, morpholine, thiomorpholine, piperazine, andhomopiperazine.

In certain preferred embodiments of formula (I), R⁵ is

wherein each R¹¹ and R¹² is independently H, halo, nitro, cyano, oroptionally fluorinated C1-C4 alkyl, optionally fluorinated C1-C4 alkoxy,COOR⁸, CONR⁹ ₂, C6-C12 aryl or C5-C12 heteroaryl, each of which may beoptionally substituted; where R⁸ is H, or C1-C8 alkyl, C2-C8 alkenyl,C6-C12 aryl, or C7-C14 arylalkyl, or a heteroform of one of these, eachof which may be optionally substituted; and

each R⁹ is independently H, or C1-C12 alkyl, C1-C12 heteroalkyl, C2-C12alkenyl, C2-C12 heteroalkenyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl,C4-C12 cycloalkylalkyl, C4-C12 heterocyclylalkyl, C6-C12 aryl, C5-C12heteroaryl, C7-C14 arylalkyl, or C6-C14 heteroarylalkyl, each of whichmay be optionally substituted; or two R⁹ on the same N can cyclize toform an optionally substituted 3- to 8-membered azacyclic ring,optionally containing an additional heteroatom selected from N, O, and Sas a ring member. Such azacyclic rings may be saturated, unsaturated oraromatic; preferred such azacyclic rings include pyrrolidine,piperidine, homopiperidine, morpholine, thiomorpholine, piperazine, andhomopiperazine.

In some such embodiments, each R¹¹ and R¹² is independently H, halo,nitro, cyano, C1-C4 alkyl, C1-C4 alkoxy, COOR⁸, or CONR⁹ ₂, C6-C12 arylor C5-C12 heteroaryl, each of which may be optionally substituted, andin particular embodiments R¹¹ is halo, nitro, cyano, C1-C4 alkyl, C1-C4alkoxy, COOR⁸, or CONR⁹ ₂, C6-C12 aryl or C5-C12 heteroaryl, each ofwhich may be optionally substituted, and R¹² is H.

Substituents on the indole and tyrosine components of the macrocylicring of formula (I), Y and Y′ respectively, are located by thecorresponding ring positions as shown in formula II:

wherein R6 is as defined in formula (I); hence, Y may be at one or moreof positions 4, 5, 6 and 7 of the indole moiety, and Y′ may be at one ormore of positions 2, 3 and 5 of the tyrosine moiety.

Preferably each Y and Y′ is independently halo (F, Cl, Br, or I), OH,C1-C4 alkoxy, preferably halo, particulary Cl or F; preferably m is 3,2, 1 or preferably, 0; and preferably m′ is 2, 1 or preferably 0.

In preferred embodiments, Y is at one or more of positions 5, 6 and 7,one or more of positions 5 and 7, one of positions 5, 6 and 7, one ofpositions 5 and 7, position 5 only, or position 7 only. In preferredembodiments, Y′ is at one or more of positions 2 and 3, one of positions2 and 3, position 2 only, or position 3 only. In particular embodimentsone or both rings are substituted.

The invention encompasses all combinations of preferred embodiments andpreferred substituents as if each had been laboriously set forth, i.e.preferred substituents at R1 combined with each preferred substituent atone or more of R2-R6 and Y/Y′/m/m′, etc. Particular examples of suchcombinations include:

Ia. Oxazole, 4 oxazoyl derviatives with esters other than methyl esterin position 4:

R¹ is C1-C4 alkyl, particulary isopropyl or t-butyl,

R², R⁴ and R⁶ are H,

R³ is a substituted methyl of the formula (—CR^(a)R^(b)R^(c)) whereinR^(a) is OH, R^(b) is H, and R^(c) is isopropyl or t-butyl,

R⁵ is

wherein R is H, C1-C4 alkyl or C1-C4 alkyloxy, particulary methyl, H, ormethoxy, andY is F and/or Cl, preferably F, at postion 5 and/or 7, preferably 5,m is 0, 1 or 2, preferably 0 or 1, andm′ is 0.

Ib. Oxazole, 4 oxazoyl derviatives with phosphate esters in position 4:

R¹ is C1-C4 alkyl, particulary isopropyl or t-butyl,

R², R⁴ and R⁶ are H,

R³ is a substituted methyl of the formula (—CR^(a)R^(b)R^(c)) whereinR^(a) is OH, R^(b) is H, and R^(c) is isopropyl or t-butyl,

R⁵ is

wherein R is H, C1-C4 alkyl, particulary methyl or H, andY is F and/or Cl, preferably F, at postion 5 and/or 7, preferably 5,m is 0, 1 or 2, preferably 0 or 1, andm′ is 0.

II. Oxazole, 4 oxazoyl derviatives with alcohol or ketone in position 4:

R¹ is C1-C4 alkyl, particulary isopropyl or t-butyl,

R², R⁴ and R⁶ are H,

R³ is a substituted methyl of the formula (—CR^(a)R^(b)R^(c)) whereinR^(a) is OH, R^(b) is H, and R^(c) is isopropyl or t-butyl,

R⁵ is

wherein R is hydroxyl or C1-C4 alcohol, or C1-C4 ketone, particularlyhydroxyl, hydroxy methyl, 1-hydroxy ethyl or 1-hydroxy isopropyl, andY is F and/or Cl, preferably F, at postion 5 and/or 7, preferably 5,m is 0, 1 or 2, preferably 0 or 1, andm′ is 0.

III. Oxazole, 4 oxazoyl derviatives with amide, amine, carbamate, orsulfonamide in position 4:

R¹ is C1-C4 alkyl, particulary isopropyl or t-butyl,

R², R⁴ and R⁶ are H,

R³ is a substituted methyl of the formula (—CR^(a)R^(b)R^(c)) whereinR^(a) is OH, R^(b) is H, and R^(c) is isopropyl or t-butyl,

R⁵ is

wherein Ra is optionally substituted C0-C4 alkyl, Rb and Rc areindependently H, C1-C8 alkyl, C2-C8 alkenyl, C6-C12 aryl, or aheteroform of one of these, each of which may be optionally substituted,particularly wherein Ra is C0 or C1 alkyl, Rb is H, and Rc is H, methyl,methyl ester, methyl sulfonyl or phenyl sulfonyl, andY is F and/or Cl, preferably F, at 5 and/or 7, preferably 5,m is 0, 1 or 2, preferably 0 or 1, andm′ is 0.

IV. Oxazole, 4 oxazoyl derviatives with cyano in position 4:

R¹ is C1-C4 alkyl, particulary isopropyl or t-butyl,

R², R⁴ and R⁶ are H,

R³ is a substituted methyl of the formula (—CR^(a)R^(b)R^(c)) whereinR^(a) is OH, R^(b) is H, and R^(c) is isopropyl or t-butyl,

R⁵ is

wherein R is H, C1-C8 alkyl, C2-C8 alkenyl, C6-C12 aryl, or a heteroformof one of these, each of which may be optionally substituted,particularly wherein R is H, methyl, or NHAc, andY is F and/or Cl, preferably F, at postion 5 and/or 7, preferably 5,m is 0, 1 or 2, preferably 0 or 1, andm′ is 0.

V. Oxazole, 4 oxazoyl derviatives with a heterocycle in position 4:

R¹ is C1-C4 alkyl, particulary isopropyl or t-butyl,

R², R⁴ and R⁶ are H,

R³ is a substituted methyl of the formula (—CR^(a)R^(b)R^(c)) whereinR^(a) is OH, R^(b) is H, and R^(c) is isopropyl or t-butyl,

R⁵ is

wherein R is a C3-C8 heterocyclyl, C4-C12 heterocyclylalkyl, C5-C12heteroaryl, or C6-C14 heteroarylalkyl, each of which may be optionallysubstituted, particularly an optionally substituted oxazole, oxazoline,thiazole, thiazoline, pyrazole, pyrazoline, imidazole, imidazoline,pyrrole, pyrroline, isoxazole, isoxazoline, isothiazole, isothiazoline,oxadiazole, thiadiazole, triazole or tetrazole ring, wherein preferredsubstituents are halo, nitro, cyano, or optionally fluorinated C1-C4alkyl, optionally fluorinated C1-C4 alkoxy, COOR^(S), CONR⁹ ₂, C6-C12aryl or C5-C12 heteroaryl, each of which may be optionally substituted;where R⁸ is H, or C1-C8 alkyl, C2-C8 alkenyl, C6-C12 aryl, or C7-C14arylalkyl, or a heteroform of one of these, each of which may beoptionally substituted; and each R⁹ is independently H, or C1-C12 alkyl,C1-C12 heteroalkyl, C2-C12 alkenyl, C2-C12 heteroalkenyl, C3-C8cycloalkyl, C3-C8 heterocyclyl, C4-C12 cycloalkylalkyl, C4-C12heterocyclylalkyl, C6-C12 aryl, C5-C12 heteroaryl, C7-C14 arylalkyl, orC6-C14 heteroarylalkyl, each of which may be optionally substituted;optionally containing an additional heteroatom selected from N, O, and Sas a ring member, andY is F and/or Cl, preferably F, at postion 5 and/or 7, preferably 5,m is 0, 1 or 2, preferably 0 or 1, andm′ is 0.

Where chiral carbons are included in chemical structures, unless aparticular orientation is depicted, both stereoisomeric forms areintended to be encompassed. Compounds of formula (I) and disclosedembodiments thereof may, for example, have two or more asymmetriccenters and therefore exist in different enantiomeric and/ordiastereomeric forms. All optical isomers and stereoisomers of thecompounds described herein, and mixtures thereof, are considered to bewithin the scope of the invention, including the racemate, one or moreenantiomeric forms, one or more diastereomeric forms, or mixturesthereof. In particular, racemic mixtures of single diastereomers such asthe ones described, diastereomers having an diastereomeric excess (d.e.)of greater than 90% or greater than about 95%, and enantiomers having anenantiomeric excess (e.e.) of greater than 90% or greater than about95%. Similarly, where double bonds are present, the compounds can existin some cases as either cis or trans isomers; the invention includeseach isomer individually as well as mixtures of isomers. Where thecompounds described may also exist in tautomeric forms, this inventionrelates to the use of all such tautomers and mixtures thereof.

Compounds of formula (I) and disclosed embodiments thereof can besupplied in free base form, or can be supplied as a pharmaceuticallyacceptable salt, or as a mixture of the free base form and thecorresponding salt. The compounds of the invention may be isolated assalts where an ionizable group such as a basic amine or a carboxylicacid is present. The invention includes the salts of these compoundsthat have pharmaceutically acceptable counterions. Such salts are wellknown in the art, and include, for example, salts of acidic groupsformed by reaction with organic or inorganic bases, and salts of basicgroups formed by reaction with organic or inorganic acids, as long asthe counterions introduced by the reaction are acceptable forpharmaceutical uses. Examples of inorganic bases with alkali metalhydroxides (e.g., sodium hydroxide, potassium hydroxide, etc.), alkalineearth metal hydroxides (e.g., of calcium, magnesium, etc.), andhydroxides of aluminum, ammonium, etc. Examples of organic bases thatcould be used include trimethylamine, triethylamine, pyridine, picoline,ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine,N,N′-dibenzylethylenediamine, etc.

Suitable salts include those of inorganic acids such as hydrochlorides,hydrobromides, sulfates, hydrosulfates, and the like, or organic acidaddition salts. Examples of inorganic acids that could be used includehydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid,phosphoric acid, etc. Examples of organic acids include formic acid,oxalic acid, acetic acid, tartaric acid, methanesulfonic acid,benzenesulfonic acid, malic acid, methanesulfonic acid, benzenesulfonicacid, p-toluenesulfonic acid, etc. Also included are salts with basicamino acids such as arginine, lysine, ornithine, etc., and salts withacidic amino acids such as aspartic acid, glutamic acid, etc.

In addition, compounds of formula (I) and disclosed embodiments thereofmay be coupled or conjugated to moieties such as a targeting agent.Among such targeting agents are antibodies or immunologically activefragments thereof, including single-chain antibody forms directedagainst tumor antigens or against receptors or integrins associated withtumors, peptidomimetics directed against these moieties, and the like.In addition, compounds of formula (I) and disclosed embodiments thereofmay be coupled or conjugated to an excipient, such as a polymerexcipient, such as polyethylene glycol for altering pharmacokinetics,such as described in the Advanced Drug Delivery Reviews theme issue (Vol61, November 2009) entitled, Polymer Therapeutics: Clinical Applicationsand Challenges for Development, including Pasut and Veronese, Adv DrugDelivery Rev 61 (13):1177-1188, 2009. The selected PEG may be of anyconvenient molecular weight, and may be linear or branched, and may beoptionally conjugated through a linker. The average molecular weight ofPEG will preferably range from about 2 kiloDalton (kDa) to about 100kDa, more preferably from about 5 kDa to about 40 kDa.

Compounds of formula (I) and disclosed embodiment thereofs are useful intreating or ameliorating cell proliferative diseases. In particular, thecompounds and methods described herein are useful for the treatment oramelioration of tumors and malignancies associated with breast, ovary,lung (SCLC and NSCLC), colon, rectum, prostate, testes, skin (e.g.,melanoma, basal cell carcinoma, and squamous cell carcinoma), pancreas,liver, kidney, brain (e.g., glioma, meningioma, schwannomas, andmedulloblastomas), and the blood and hematopoietic system, including,e.g., leukemia, non-Hodgkins lymphoma, and multiple myeloma.

In the methods described herein, for example, cell proliferation may bereduced, and/or cell death, such as apoptosis or apoptotic cell death,may be induced. The cell proliferative disorder may be a tumor ornon-tumor cancer in a human or animal subject.

The compounds and methods provided herein for reducing cellproliferation and/or inducing cell death may be used alone, or inconjunction with or in combination with surgical, radiation,chemotherapeutic, immunotherapy, and bone marrow and/or stem celltransplantation methods, or with other palliative agents, such ascompounds that aid in nutrition or general health, anti-emetic agents,and the like.

In some embodiments, the compounds of the present invention areadministered in combination with a chemotherapeutic agent, and used toreduce cell proliferation, induce cell death, and/or treat or amelioratea cell proliferative disorder.

The compounds described herein are also useful against certain drugresistant tumors and cancer cell lines, in particular against cancersthat are resistant to TAXOL® and/or vinca alkaloid anti-cancer agents.

Where an additional chemotherapeutic drug is administered, it istypically one known to have cytostatic, cytotoxic or antineoplasticactivity. These agents include, without limitation, antimetabolites(e.g., cytarabine, fludaragine, 5-fluoro-2′-deoxyuridine, gemcitabine,hydroxyurea, methotrexate); DNA active agents (e.g., bleomycin,chlorambucil, cisplatin, cyclophosphamide); intercalating agents (e.g.,adriamycin and mitoxantrone); protein synthesis inhibitors (e.g.,L-asparaginase, cycloheximide, puromycin); topoisomerase type Iinhibitors (e.g., camptothecin, topotecan or irinotecan); topoisomerasetype II inhibitors (e.g. etoposide, teniposide anthraquinones,anthracyclines and podophyllotoxin); microtubule inhibitors (e.g.,taxanes, such as paclitaxel and docetaxel, colcemid, colchicines, orvinca alkaloids, such as vinblastine and vincristine); kinase inhibitors(e.g. flavopiridol, staurosporin and hydroxystaurosporine), drugs thataffect Hsp90 (e.g. geldanomycin and geldanomycin derivatives, radicicol,purine derivatives and antibodies or antibody fragments that selectivelybind to Hsp90), TRAIL, a TRAIL receptor antibody, TNF-α or TNF-β, and/orradiation therapy.

In some preferred embodiments, the additional cancer therapeutic agentis TRAIL, a TRAIL receptor antibody, TNF-α or TNF-β. In other preferredembodiments, the additional drugs for co-administration with thecompounds of the invention affects Hsp90 (heat-shock protein 90).

Suitable Hsp90 inhibitors include ansamycin derivatives such asgeldanomycin and geldanomycin derivatives including17-(allylamino)-17-desmethoxygeldanamycin (17-AAG), its dihydroderivative, 17-AAGH₂, and 17-amino derivatives of geldanamycin such as17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG),11-oxogeldanamycin, and 5,6-dihydrogeldanamycin, which are disclosed inU.S. Pat. Nos. 4,261,989; 5,387,584; and 5,932,566, each of which isincorporated herein by reference. Other suitable Hsp90 inhibitorsinclude radicicol and oximes and other analogs thereof, disclosed inSoga, et al., Curr. Cancer Drug Targets, 3, 359-69 (2003), and inYamamoto, et al., Angew. Chem., 42, 1280-84 (2003); and in Moulin, etal., J. Amer. Chem. Soc., vol 127, 6999-7004 (2005); purine derivativessuch as PU3, PU24FCI and PUH64 (see Chiosis et al., ACS Chem. Biol. Vol.1(5), 279-284 (2006) and those disclosed in PCT Application No. WO2002/0236075; related heterocyclic derivatives disclosed in PCTApplication No. WO 2005/028434; and 3,4-diarylpyrazole compoundsdisclosed in Cheung, et al., Bioorg. Med. Chem. Lett., vol. 15, 3338-43(2005). Antibodies or antibody fragments that selectively bind to Hsp90may also be administered as drugs to cause inhibition of Hsp90, and canbe used in combination with the compounds of the invention.

Where a compound described herein is utilized in conjunction with or incombination with another therapeutic, the two agents may beco-administered, or they may be administered separately where theiradministration is timed so the two agents act concurrently orsequentially.

Accordingly, the compositions used in the methods described hereininclude at least one compound of the invention, and can optionallyinclude one or more additional cytotoxic or cytostatic therapeutic suchas, but not limited to, those disclosed above. Similarly, the methods ofthe invention include methods wherein a subject diagnosed as in need oftreatment for cancer is treated with at least one compound orcomposition of the invention, and is simultaneously or concurrentlytreated with one or more of the additional therapeutic agents describedabove.

Formulation and Administration

The formulations useful in the invention include standard formulationssuch as those set forth in Remington's Pharmaceutical Sciences, latestedition, Mack Publishing Co., Easton, Pa., incorporated herein byreference. Such formulations include those designed for oral delivery,slow release, topical administration, parenteral administration, or anyother suitable route as determined by an attending physician orveterinarian. Thus administration may be systemic or local. Suitablevehicles or excipients include liposomes, micelles, nanoparticles,polymeric matrices, buffers, and the full range of formulations known topractitioners.

Systemic formulations include those designed for injection (e.g.,intramuscular, intravenous or subcutaneous injection) and those preparedfor transdermal, transmucosal, or oral administration. The formulationwill generally include a diluent as well as, in some cases, adjuvants,buffers, preservatives and the like. The compounds can be administeredalso in liposomal compositions or as microemulsions.

Injection methods are sometimes appropriate routes for administration ofthe compounds for systemic treatments and sometimes also for localizedtreatments. These include methods for intravenous, intramuscular,subcutaneous, and other methods for internal delivery that bypass themucosal and dermal barriers to deliver the composition directly into thesubject's living tissues.

For injection, formulations can be prepared in conventional forms asliquid solutions or suspensions or as solid forms suitable for solutionor suspension in liquid prior to injection or as emulsions. Suitableexcipients include, for example, water, saline, dextrose, glycerol andthe like. Such compositions may also contain amounts of nontoxicauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and the like, such as, for example, sodium acetate, sorbitanmonolaurate, and so forth.

Various sustained release systems for drugs have also been devised andcan be utilized with the compounds of the invention. See, for example,U.S. Pat. No. 5,624,677. The present compositions can be utilized insuch controlled-release delivery systems where appropriate.

Systemic administration may also include relatively noninvasive methodssuch as the use of suppositories, transdermal patches, transmucosaldelivery and intranasal administration. Oral administration is alsosuitable for compounds of the invention. Suitable forms include syrups,capsules, tablets, and the like as in understood in the art.

Selection of a particular route of administration for a given subjectand indication is well within the ordinary level of skill in the art.For example, rectal delivery as a suppository is often appropriate wherethe subject experiences nausea and vomiting that precludes effectiveoral delivery. Transdermal patches are commonly capable of delivering acontrolled-release dosage over several days or to a specific locus, andare thus suitable for subjects where these effects are desired.

Transmucosal delivery is also appropriate for some of the compositionsand methods of the invention. Thus the compositions of the invention maybe administered transmucosally using technology and formulation methodsthat are known in the art.

Regardless of the route of administration selected, the compoundsdescribed herein, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

For administration to animal or human subjects, the dosage of a compoundof the invention is typically 10-2400 mg per administration. However,dosage levels are highly dependent on the nature of the condition, thecondition of the patient, the judgment of the practitioner, and thefrequency and mode of administration. Selection of a dosage of suchcompounds is within the skill of an ordinary artisan, and may beaccomplished by starting at a relatively low dosage and increasing thedosage until an acceptable effect is achieved.

Frequency of administration of the compounds of the invention can alsobe readily determined by one skilled in the art using well knowntechniques. For example, the patient may be administered a low dosage ofa compound or composition of the invention at a low frequency such asonce per day or less often; and the dosage and/or frequency ofadministration may be systematically increased until a desired effect isachieved in the patient.

Synthetic Processes

The subject compounds have been prepared through an efficient multi-stepprocess, as shown in Scheme 1. A key step in the process involves theelectrochemical oxidative cyclization of a phenolic intermediate toprovide an indoline compound of formula (I), which may be furtherfunctionalized as exemplified by the compounds described herein. Theoxidative cyclization was described in U.S. application Ser. No.12/134,984, filed 6 Jun. 2008, and published as US 2009/0005572.

As shown in Scheme 1, dipeptide starting materials were prepared understandard conditions known in the art, for example, by coupling anN-hydroxysuccinimide ester or another activated ester of a protectedamino acid with serine. It will be understood by one of skill in the artthat a wide variety of suitable conditions may be utilized to form thedipeptide starting materials, including the extensive body of literaturedescribing synthesis of peptides and peptide mimetics.

The dipeptide was reacted with an optionally substituted indole and anactivating reagent, optionally in the presence of a protic acid, toprovide an indole-containing dipeptide. Suitable activating reagentsinclude, for example, carboxylic acid anhydrides, mixed anhydrides, oracyl halides (e.g., acetic anhydride, trifluoroacetic anhydride, acetylchloride, oxalyl chloride), sulfonic acid anhydrides or halides (e.g.,methanesulfonic anhydride, trifluoromethanesulfonic anhydride,methanesulfonyl chloride), mineral acid halides (e.g., thionyl chloride,or phosphoryl chloride), and the like.

In a preferred embodiment, the activating agent was acetic anhydride,and the reaction was conducted in acetic acid as a protic solvent. In aparticularly preferred embodiment, the dipeptide and an optionallysubstituted indole were reacted with acetic anhydride in acetic acid atabout 80° C., to provide the desired compound.

The preparation of N-acetyl tryptophan derivatives by reaction of serineor N-acetyl serine and an optionally substituted indole in aceticanhydride and acetic acid has been previously reported. Y. Yokoyama, etal., Tetrahedron Letters (1999), 40: 7803; Y. Yokoyama, et al., Eur. J.Org. Chem. (2004), 1244; Y. Konda-Yamada, et al., Tetrahedron (2002),58: 7851; M. W. Orme, et al., U.S. Pat. No. 6,872,721. However, thepreparation of other acylated tryptophan derivatives under theseconditions, such as the dipeptide analogs of the present invention, hasnot been previously described to our knowledge.

Esterification of the free carboxylic acid, followed by oxidativecyclization of the dipeptide intermediate with an oxidizing agent, forexample, DDQ, provided an oxazole intermediate. It will be understood bythose in the art that other oxidative conditions could be utilized, suchas, for example, the use of 7,7,8,8-tetracyanoquinodimethane (TCNQ),ceric ammonium nitrate, hypervalent iodide reagents, and the like.

Deprotection of the protected amino group, if present, and amide bondformation provided a phenolic intermediate. Electrochemical oxidativecyclization of the phenolic intermediate provided a macrocyclic indolinecompound. Such compounds were further elucidated to compounds of formula(I) through a series of straightforward chemical transformation. Forexample, removal of the Cbz group and acylation or amide bond formationwas used to provide compounds of formula (I), wherein R⁵ is an acylsubstituent, for example —C(O)R³. One of skill in the art willunderstand that the order of these steps could be reversed, depending onthe nature of the functional groups to be installed, and the protectinggroups utilized.

Scheme 1 provides a general synthetic route useful for the preparationof macrocyclic indoline compounds of formula (I). Those skilled in theart will appreciate that certain reaction conditions can be variedwithout altering the essence of the present invention. For example,coupling reactions can be accomplished with a variety of activatedesters, such as by way of example only N-hydroxybenzotriazole ester,perfluorophenyl ester, N-hydroxyphthalimide esters, activated estersgenerated by the reaction of the carboxylic acid with a carbodiimide,and other activated esters conventionally used for acylation of an amineto form amide bonds. In addition, while amino groups are convenientlyprotected as carbobenzyloxy (Cbz) group, one of skill in the art willrecognize that other suitable protecting groups could be utilized.Suitable protecting groups and methods to attach and remove them arewell known in the art, and are described, for example, in T. H. Greene,Protective Groups in Organic Synthesis, 2^(nd) ed.

The process described in Scheme 1 is useful for the preparation ofindolines of formula (I) in high yield and purity. In particular, thecompounds of the present invention are available in good yield and withhigh diastereomeric purity, preferably in greater than 95%diastereomeric excess, sometimes 98% diastereomeric excess.

The following examples are offered to illustrate but not to limit theinvention.

EXAMPLES Synthesis of Compound 57

Step 1

To a dry 100-ml flask with magnetic stir bar was addedCbz-L-α-t-butylglycine DCHA salt (5.0 g, 11.2 mmol), L-tryptophanemethyl ester hydrochloride (3.14 g, 12.3 mmol, 1.1 eq.), HOBt (1.76 g,13.4 mmol, 1.2 eq.), anhydrous DMF (30 ml) and N,N-diisopropylethylamine(2.93 ml, 16.8 mmol, 1.5 eq.). The reaction mixture was cooled to 0° C.followed by addition of EDC HCl (2.58 g, 13.4 mmol, 1.2 eq.). Theresulting reaction mixture was stirred at RT for 16 h. The reaction wasmonitored by LCMS. The reaction mixture was diluted with EtOAc (300ml)/water (100 ml). The organic phase was separated and the aqueousphase was extracted by EtOAc (2×50 ml). The combined organic layers werewashed by water (100 ml), 10% aqueous NaHSO₄ (100 ml), water (100 ml),saturated NaHCO₃ (100 ml), and brine (2×100 ml), and then dried overNa₂SO₄. After concentration, the crude was used directly in the nextstep.

Step 2

A solution of DDQ (6.2 g, 27.3 mmol, 2.4 eq.) in THF (100 ml) was addedto the refluxing solution of the compound synthesized in Step 1 above(11.2 mmol) in THF (200 ml) and the dark solution was heated in refluxin an oil bath at 85° C. for 1 h. After cooling, the solvent was removedon a rotary evaporator. The residue was dissolved in ethyl acetate (500ml), which was washed by water (200 ml), aqueous saturated NaHCO₃ (2×200ml), water (2×200 ml), brine (100 ml) and dried over Na₂SO₄. Afterconcentration, the mixture was purified by flash column chromatography(20% EtOAc in CH₂Cl₂). This yielded 3.24 g (64% yield) of product.

Step 3

To a 100-ml flask containing material synthesized in Step 2 above (3.24g, 7.02 mmol) was added methanol (30 ml) and Pd/C (10%) (650 mg, 0.61mmol, 0.09 eq.) under N₂. H₂ balloon was added and the flask was purgedwith H₂ for 4 times. Then H₂ balloon was opened to the reaction system.After 3 h stirring almost no starting material remained. The reactionwas stopped. The reaction mixture was filtered through a pad of Celiteand the black cake was washed with methanol (3×10 ml). The filtrate wasconcentrated and the residue was used in next step directly withoutfurther purification.

Step 4

To a dry 100-ml flask with magnetic stir bar was added the aminesynthesized in step 3 (2.06 g, 6.29 mmol), Cbz-L-tyrosine (1.98 g, 6.91mmol, 1.1 eq.), HOBt (0.94 g, 6.91 mmol, 1.1 eq.), anhydrous DMF (30 ml)and N,N-diisopropylethylamine (1.31 ml, 7.54 mmol, 1.2 eq.). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(1.33 g, 6.91 mmol, 1.1 eq.). The resulting reaction mixture was stirredat RT for 16 h. The reaction was monitored by LCMS. The reaction mixturewas diluted with EtOAc (300 ml)/water (100 ml). The organic phase wasseparated and the aqueous phase was extracted by EtOAc (2×50 ml). Thecombined organic layers were washed by water (100 ml), 10% aqueousNaHSO₄ (100 ml), water (100 ml), saturated NaHCO₃ (100 ml), and brine(2×100 ml), and then dried over Na₂SO₄. After concentration, the crudewas used directly in the next step.

Step 5

An electrochemical cell was assembled using a glass cylinder (6 cmdiameter×11 cm height) and a custom rack (polypropylene and nylon) whichsupported 9 vertical graphite rods (6.15 mm diameter×12 cm length). Therods were arranged in a pattern of a ring with 6 anodes and 3 cathodes.Electrodes were immersed to a depth of 6.5 cm. The phenolic materialsynthesized in Step 4 above (5.00 g, 8.0 mmol), Et₄NBF₄ (4.00 g, 18.4mmol, 2.9 eq.) and (NH₄)₂CO₃(1.0 g, 10.4 mmol, 1.3 eq.) and ID water (4ml) were added in DMF (200 ml). The solution was stirred vigorously in astir plate (approx. 600 rpm). The electrochemical reaction was carriedout at a potential of 1.5-1.6 volts. After 3 days, most of the originalSM was consumed as determined by HPLC integration at 220 nM. Thereaction mixture was concentrated on a rotary evaporator (bath temp.≦35° C.) and dried further on a vacuum manifold. The residue waspartitioned between EtOAc (200 ml) and 0.5 N aqueous HCl (60 ml). Theorganic layer was washed with saturated aqueous NaHCO₃ (50 ml) and thensaturated aqueous NaCl (50 ml). The aqueous layers were extracted insuccession with EtOAc (2×50 ml). The combined organic layers were dried(Na₂SO₄), decanted and evaporated. This material was purified by flashcolumn chromatography with 20% EtOAc in CH₂Cl₂. This yielded 1.24 g(24.8% yield) of product as a mixture of stereoisomers (71:29 asmeasured by HPLC integration at 254 nM).

Step 6

The compound synthesized in Step 6 (725 mg, 2.33 mmol) was dissolved inmethanol (22 ml) and the solution was cooled in an ice bath. A solutionof LiOH (558 mg, 23.3 mmol, 10 eq.) in water (7.0 ml) was added over 5min. The ice bath was removed and the mixture was stirred for 18 h. Themixture was cooled in an ice bath and water (30 ml) was added followedby 1 N aqueous HCl (24 ml), keeping the reaction temperature below 10°C. The mixture was partitioned between water (15 ml) and EtOAc (100 ml),and the organic layer was washed with saturated aqueous NaCl. Theaqueous layers were extracted in succession with EtOAc (30 ml). Thecombined organic layers were dried (Na₂SO₄), decanted, and evaporated togive the acid product as fine white crystals.

Step 7

To a dry 100-ml flask with magnetic stir bar was added the carboxylicacid synthesized in step 6 above (2.33 mmol), L-serine methyl esterhydrochloride (435 mg, 2.8 mmol, 1.2 eq.), HOBt (378 mg, 2.8 mmol, 1.2eq.), anhydrous DMF (25 ml) and N,N-diisopropylethylamine (1.01 ml, 5.83mmol, 2.5 eq.). The reaction mixture was cooled to 0° C. followed byaddition of EDC HCl (537 mg, 2.8 mmol, 1.2 eq.). The resulting reactionmixture was stirred at RT for 16 h. The reaction was monitored by LCMS.Most of solvents were evaporated under reduced pressure. The residue wasdiluted with EtOAc (100 ml)/water (30 ml). The organic phase wasseparated and the aqueous phase was extracted by EtOAc (2×20 ml). Thecombined organic layers were washed by water (40 ml), 10% aqueous NaHSO₄(40 ml), water (40 ml), saturated NaHCO₃ (40 ml), and brine (2×40 ml),and then dried over Na₂SO₄. After concentration, the crude was useddirectly in the next step.

Step 8

To a dry flask were added the crude product from Step 8 above (2.33mmol) and anhydrous CH₂Cl₂ (40 ml). The reaction solution became cloudyas it was cooled to −20° C. in a dry ice/acetone/water bath. A freshlymade stock solution of Bis(2-methoxyethyl)aminosulfur trifluoride (0.644ml, 0.022 mmol, 2.8 eq.) in CH₂Cl₂ (4 ml) was added dropwise. Theresulting reaction mixture was stirred at −20° C. for 1 h, and warmed toroom temperature. The reaction mixture was quenched by addition ofsaturated aqueous NaHCO₃ (20 ml), diluted with EtOAc (100 ml), washedwith water (2×30 ml) as well as brine (30 ml), and dried over Na₂SO₄.After concentration the residue was used in next step.

Step 9

To a dry flask containing the crude product from step 8 above (2.33mmol) were added anhydrous CH₂Cl₂ (40 ml). The mixture was cooled to 0°C. Then CBrCl₃ (0.345 ml, 3.5 mmol, 1.5 eq.) and DBU (0.523 ml, 3.5mmol, 1.5 eq.) were added respectively. The resulting mixture wasallowed to warm to room temperature and was stirred for 1 h. Thereaction was monitored by LCMS. The reaction mixture was diluted withEtOAc (100 ml), washed by 10% NaHSO₄ (30 ml), water (2×30 ml), saturatedaqueous NaHCO₃ (30 ml), water (30 ml) and brine (30 ml), dried overNa₂SO₄. After concentration the residue was used in next step.

Step 10

To a 50-ml flask containing material synthesized in Step 9 above (400mg, 0.58 mmol) were added methanol (15 ml), t-butylamine (0.086 ml, 0.87mmol, 1.5 eq.) and Pd/C (10%) (62 mg, 0.058 mmol, 0.1 eq.) under N₂. H₂balloon was added and the flask was purged with H₂ for 4 times. Then H₂balloon was opened to the reaction system. After 4 h stirring almost nostarting material remained. The reaction was stopped. The reactionmixture was filtered through a pad of Celite and the black cake waswashed with methanol (3×10 ml). The filtrate was concentrated and theresidue was used in next step directly without further purification.

Step 11

To a dry 25-ml flask containing the amine synthesized in Step 10 above(0.58 mmol) were added (S)-(+)-2-Hydroxy-3-methylbutanoic acid (82 mg,0.696 mmol, 1.2 eq.), HOB t (94 mg, 0.696 mmol, 1.2 eq.), anhydrous DMF(8 ml) and N,N-diisopropylethylamine (0.152 ml, 0.87 mmol, 1.5 eq.). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(133 mg, 0.696 mmol, 1.2 eq.). The resulting reaction mixture wasstirred at room temperature for 16 h. The reaction was monitored byLCMS. The reaction mixture was diluted with EtOAc (80 ml)/water (30 ml).The organic phase was separated and the aqueous phase was extracted byEtOAc (2×20 ml). The combined organic layers were washed by water (30mL), 10% aqueous NaHSO₄ (30 ml), water (30 ml), saturated NaHCO₃ (30ml), and brine (2×30 ml), and then dried over Na₂SO₄. Afterconcentration, the crude was used directly in the next step.

Step 12

To a dry flask were added crude material synthesized in Step 11 (0.58mmol), THF (4 ml) and 2-propanol (12 ml). This solution was cooled to 0°C. followed by addition of solid lithium borohydride (152 mg, 6.96 mmol,12 eq.). The resulting mixture was allowed to warm to room temperatureand stirred for 22 h. The reaction was monitored with LCMS. Almost nostarting material remained. The reaction mixture was cooled to 0° C.2-Propanol (24 ml) and water (40 ml) were added followed by addition ofNH₄Cl (3.1 g, 58 mmol, 100 eq.). The reaction mixture was stirred for 1h and diluted with EtOAc (250 ml)/water (50 ml). The organic phase wasseparated and the aqueous phase was extracted by EtOAc (2×50 ml). Thecombined organic layers were washed by water (3×50 ml), 10% NaHSO₄ (2×50ml), water (2×50 ml), saturated NaHCO₃ (50 ml), and brine (2×50 ml), andthen dried over Na₂SO₄. After concentration the residue was purified byflash column chromatography (EtOAc to 10% EtOAc/MeOH) to afford desiredproduct as an off-white solid (188 mg, 0.108 mmol, 52% for three steps).MS: m/z=627.9 (M+1).

Synthesis of Compound 81

Step 1

To a dry 250-ml flask were added 5-fluoro-DL-tryptophane (5.0 g, 22.5mmol), and anhydrous methanol (120 ml). The suspension was cooled to 0°C. followed by addition of chlorotrimethyl silane (12.8 ml, 101.3 mmol,4.5 eq.) in such a rate to keep the reaction temperature below 6° C. Theresulting reaction mixture was stirred at room temperature for 20 h. Thereaction was monitored by TLC. Most volatile substances were evaporatedunder reduced pressure. The crude was used in next step.

Step 2

To a dry 250-ml flask with magnetic stir bar was added the amine saltsynthesized in step 1 above (22.5 mmol.), Cbz-L-valine (6.22 g, 24.75mmol, 1.1 eq.), HOB t (3.34 g, 24.75 mmol, 1.1 eq.), anhydrous DMF (80ml) and N,N-diisopropylethylamine (11.8 ml, 67.5 mmol, 3.0 eq.). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(4.74 g, 24.75 mmol, 1.1 eq.). The resulting reaction mixture wasstirred at RT for 16 h. The reaction was monitored by LCMS. Most ofsolvents were evaporated under reduced pressure. Then the residue wasdiluted with EtOAc (600 ml)/water (200 ml). The organic phase wasseparated and the aqueous phase was extracted by EtOAc (2×50 ml). Thecombined organic layers were washed by water (100 ml), 10% aqueousNaHSO₄ (100 ml), water (100 ml), saturated NaHCO₃ (100 ml), and brine(2×100 ml), and then dried over Na₂SO₄. After concentration, the crudewas used directly in the next step.

Step 3

A solution of DDQ (12.8 g, 56.25 mmol, 2.5 eq.) in THF (500 ml) wasadded to the refluxing solution of the compound synthesized in Step 2above (22.5 mmol) in THF (250 ml) and the dark solution was kept inreflux in an oil bath at 85° C. for 1 h. After cooling, the solvent wasremoved on a rotary evaporator. The residue was dissolved in ethylacetate (600 ml), and NaHCO₃ (13 g) was added. The mixture was stirredfor 1 h followed by filtration through a fritted funnel. The filtratewas washed by water (200 ml), aqueous saturated NaHCO₃ (2×200 ml), water(2×200 ml), brine (100 ml) and dried over Na₂SO₄. After concentration,the mixture was purified by flash column chromatography (5% EtOAc inCH₂Cl₂). This yielded 4.63 g (44.2% yield) of product.

Step 4

To a 250-ml flask containing material synthesized in Step 3 above (4.63g, 9.94 mmol) was added methanol (50 ml) and Pd/C (10%) (530 mg, 0.497mmol, 0.05 eq.) under N₂. H₂ balloon was added and the flask was purgedwith H₂ for 4 times. Then H₂ balloon was opened to the reaction system.After 1 h stirring almost no starting material remained. The reactionwas stopped. The reaction mixture was filtered through a pad of Celiteand the black cake was washed with methanol (3×15 ml). The filtrate wasconcentrated and the residue was used in next step directly withoutfurther purification.

Step 5

To a dry 100-ml flask with magnetic stir bar was added the aminesynthesized in step 4 (9.94 mmol), Cbz-L-tyrosine (3.45 g, 10.93 mmol,1.1 eq.), HOBt (1.48 g, 10.93 mmol, 1.1 eq.), anhydrous DMF (30 ml). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(2.10 g, 10.93 mmol, 1.1 eq.). The resulting reaction mixture wasstirred at RT for 16 h. The reaction was monitored by LCMS. The reactionmixture was diluted with EtOAc (400 ml)/water (150 ml). The organicphase was separated and the aqueous phase was extracted by EtOAc (2×100ml). The combined organic layers were washed by water (200 ml), 10%aqueous NaHSO₄ (150 ml), water (150 ml), saturated NaHCO₃ (150 ml), andbrine (2×100 ml), and then dried over Na₂SO₄. After concentration, thecrude (6.58 g) was used directly in the next step.

Step 6

An electrochemical cell was assembled using a glass cylinder (6 cmdiameter×11 cm height) and a custom rack (polypropylene and nylon) whichsupported 9 vertical graphite rods (6.15 mm diameter×12 cm length). Therods were arranged in a pattern of a ring with 6 anodes and 3 cathodes.Electrodes were immersed to a depth of 6.5 cm. The phenolic materialsynthesized in Step 5 above (2.00 g, 3.18 mmol), Et₄NBF₄ (2.00 g, 9.2mmol, 3 eq.), K₂CO₃(0.44 g, 3.18 mmol, 1.0 eq.) and ID water (4 ml) wereadded in DMF (200 ml). The solution was stirred vigorously in a stirplate (approx. 600 rpm). The electrochemical reaction was carried out ata potential of 1.5-1.6 volts. After 3 days, most of the original SM wasconsumed as determined by HPLC integration at 220 nM. Theelectrochemistry reaction was repeated for 4 times to consume allphenolic material synthesized in step 5. The combined reaction mixtureswere concentrated on a rotary evaporator (bath temp. ≦35° C.) and driedfurther on a vacuum manifold. The residue was diluted with EtOAc (500ml) followed by filtration through a fritted funnel. The filtrate waswashed by water (2×200 ml), brine (200 ml). The aqueous layers wereextracted in succession with EtOAc (2×50 ml). The combined organiclayers were dried (Na₂SO₄) and concentrated. This material was purifiedby flash column chromatography with 15% MeCN in CH₂Cl₂. This yielded 900mg of desired product with 14% yield in three steps.

Step 7

The compound synthesized in Step 6 (900 mg, 1.43 mmol) was dissolved inmethanol (28 ml) and the solution was cooled in an ice bath. A solutionof LiOH (344 mg, 14.3 mmol, 10 eq.) in water (4.5 ml) was added over 5min. The ice bath was removed and the mixture was stirred at RT for 18h. The mixture was cooled in an ice bath and water (40 ml) was addedfollowed by 1 N aqueous HCl (14.5 ml), keeping the reaction temperaturebelow 10° C. The mixture was partitioned between water (25 ml) and EtOAc(200 ml), and the organic layer was washed with saturated aqueous NaCl.The aqueous layers were extracted in succession with EtOAc (50 ml). Thecombined organic layers were dried (Na₂SO₄), decanted, and evaporated togive the acid product as fine white crystals.

Step 8

To a dry 50-ml flask with magnetic stir bar was added the carboxylicacid synthesized in step 7 above (1.43 mmol), L-serine methyl esterhydrochloride (268 mg, 1.72 mmol, 1.2 eq.), HOBt (232 mg, 1.72 mmol, 1.2eq.), anhydrous DMF (15 ml) and N,N-diisopropylethylamine (0.624 ml,3.58 mmol, 2.5 eq.). The reaction mixture was cooled to 0° C. followedby addition of EDC HCl (330 mg, 1.72 mmol, 1.2 eq.). The resultingreaction mixture was stirred at RT for 16 h. The reaction was monitoredby LCMS. Most of solvents were evaporated under reduced pressure. Theresidue was diluted with EtOAc (150 ml)/water (50 ml). The organic phasewas separated and the aqueous phase was extracted by EtOAc (2×30 ml).The combined organic layers were washed by water (60 ml), 10% aqueousNaHSO₄ (60 ml), water (60 ml), saturated NaHCO₃ (60 ml), and brine (2×60ml), and then dried over Na₂SO₄. After concentration, the crude was useddirectly in the next step.

Step 9

To a dry flask were added the crude product from Step 8 above (1.43mmol) and anhydrous CH₂Cl₂ (25 ml). The reaction solution became cloudyas it was cooled to −20° C. in a dry ice/acetone/water bath. A freshlymade stock solution of Bis(2-methoxyethyl)aminosulfur trifluoride (0.395ml, 2.15 mmol, 1.5 eq.) in CH₂Cl₂ (4 ml) was added dropwise. Theresulting reaction mixture was stirred at −20° C. for 1 h, and warmed toroom temperature. The reaction mixture was quenched by addition ofsaturated aqueous NaHCO₃ (15 ml), diluted with EtOAc (100 ml), washedwith water (2×20 ml) as well as brine (30 ml), and dried over Na₂SO₄.After concentration the residue was used in next step.

Step 10

To a dry flask containing the crude product from step 9 above (1.43mmol) were added anhydrous CH₂Cl₂ (25 ml). The mixture was cooled to 0°C. Then CBrCl₃ (0.211 ml, 2.15 mmol, 1.5 eq.) and DBU (0.321 ml, 2.15mmol, 1.5 eq.) were added respectively. The resulting mixture wasallowed to warm to room temperature and was stirred for 1 h. Thereaction was monitored by LCMS. The reaction mixture was diluted withEtOAc (100 ml), washed by 10% NaHSO₄ (30 ml), water (2×30 ml), saturatedaqueous NaHCO₃ (15 ml), water (30 ml) and brine (30 ml), dried overNa₂SO₄. After concentration the residue was used in next step.

Step 11

To a 100-ml flask containing material synthesized in Step 10 above (1.43mmol) were added methanol (20 ml), t-butylamine (0.226 ml, 2.15 mmol,1.5 eq.) and Pd/C (10%) (152 mg, 0.143 mmol, 0.1 eq.) under N₂. H₂balloon was added and the flask was purged with H₂ for 4 times. Then H₂balloon was opened to the reaction system. After 4 h stirring almost nostarting material remained. The reaction was stopped. The reactionmixture was filtered through a pad of Celite and the black cake waswashed with methanol (3×15 ml). The filtrate was concentrated and theresidue was used in next step directly without further purification.

Step 12

To a dry 25-ml flask containing the amine synthesized in Step 11 above(1.43 mmol) were added (S)-(+)-2-hydroxy-3-methylbutanoic acid (203 mg,1.72 mmol, 1.2 eq.), HOBt (232 mg, 1.72 mmol, 1.2 eq.), anhydrous DMF(15 ml) and N,N-diisopropylethylamine (0.374 ml, 2.15 mmol, 1.5 eq.).The reaction mixture was cooled to 0° C. followed by addition of EDC HCl(330 mg, 1.72 mmol, 1.2 eq.). The resulting reaction mixture was stirredat room temperature for 16 h. The reaction was monitored by LCMS. Thereaction mixture was diluted with EtOAc (150 ml)/water (50 ml). Theorganic phase was separated and the aqueous phase was extracted by EtOAc(2×30 ml). The combined organic layers were washed by water (50 ml), 10%aqueous NaHSO₄ (50 ml), water (30 ml), saturated NaHCO₃ (50 ml), andbrine (2×50 ml), and then dried over Na₂SO₄. After concentration, thecrude was used directly in the next step.

Step 13

To a dry flask were added crude material synthesized in Step 12 (1.43mmol), THF (13 ml) and 2-propanol (40 ml). This solution was cooled to0° C. followed by addition of solid lithium borohydride (467 mg, 21.45mmol, 15 eq.). The resulting mixture was allowed to warm to roomtemperature and stirred for 18 h. The reaction was monitored with LCMS.Almost no starting material remained. The reaction mixture was cooled to0° C. 2-Propanol (40 ml) and water (80 ml) were added followed byaddition of NH₄Cl (7.6 g, 143 mmol, 100 eq.). The reaction mixture wasstirred for 1 h and diluted with EtOAc (400 ml)/water (100 ml). Theorganic phase was separated and the aqueous phase was extracted by EtOAc(2×100 ml). The combined organic layers were washed by water (3×100 ml),10% NaHSO₄ (2×100 ml), water (2×100 ml), saturated NaHCO₃ (100 ml), andbrine (2×100 ml), and then dried over Na₂SO₄. After concentration theresidue was purified by flash column chromatography (Pure EtOAc to 7%MeCN/EtOAc) to afford desired product as an off-white solid (215 mg,0.340 mmol, 24% for seven steps).

Synthesis of Compound 85

Step 1

To a dry 250-ml flask were added 5-fluoro-DL-tryptophane (6.0 g, 27.0mmol), and anhydrous methanol (120 ml). The suspension was cooled to 0°C. followed by addition of chlorotrimethyl silane (15.4 ml, 121.5 mmol,4.5 eq.) in such a rate to keep the reaction temperature below 6° C. Theresulting reaction mixture was stirred at room temperature for 20 h. Thereaction was monitored by TLC. Most volatile substances were evaporatedunder reduced pressure. The crude was used in next step.

Step 2

To a dry 250-ml flask with magnetic stir bar was added the amine saltsynthesized in step 1 above (27 mmol.), Cbz-L-α-t-butylglycine DCHA salt(13.26 g, 29.7 mmol, 1.1 eq.), HOBt (4.01 g, 29.7 mmol, 1.1 eq.),anhydrous DMF (100 ml) and N,N-diisopropylethylamine (14.1 ml, 81 mmol,3.0 eq.). The reaction mixture was cooled to 0° C. followed by additionof EDC HCl (5.69 g, 29.7 mmol, 1.1 eq.). The resulting reaction mixturewas stirred at RT for 16 h. The reaction was monitored by LCMS. Most ofsolvents were evaporated under reduced pressure. Then the residue wasdiluted with EtOAc (700 ml)/water (200 ml). The organic phase wasseparated and the aqueous phase was extracted by EtOAc (2×50 ml). Thecombined organic layers were washed by water (100 ml), 10% aqueousNaHSO₄ (100 ml), water (100 ml), saturated NaHCO₃ (100 ml), and brine(2×100 ml), and then dried over Na₂SO₄. After concentration, the crudewas used directly in the next step.

Step 3

A solution of DDQ (15.32 g, 67.5 mmol, 2.5 eq.) in THF (100 ml) wasadded to the refluxing solution of the compound synthesized in Step 2above (27 mmol) in THF (300 ml) and the dark solution was kept in refluxin an oil bath at 85° C. for 1 h. After cooling, the solvent was removedon a rotary evaporator. The residue was dissolved in ethyl acetate (700ml), and NaHCO₃ (15 g) was added. The mixture was stirred for 1 hfollowed by filtration through a fritted funnel. The filtrate was washedby water (200 ml), aqueous saturated NaHCO₃ (2×200 ml), water (2×200ml), brine (100 ml) and dried over Na₂SO₄. After concentration, themixture was purified by flash column chromatography (5% EtOAc inCH₂Cl₂). This yielded 6.42 g (50% yield) of product.

Step 4

To a 250-ml flask containing material synthesized in Step 3 above (6.42g, 13.4 mmol) was added methanol (60 ml) and Pd/C (10%) (1.43 g, 1.34mmol, 0.1 eq.) under N₂. H₂ balloon was added and the flask was purgedwith H₂ for 4 times. Then H₂ balloon was opened to the reaction system.After 1 h stirring almost no starting material remained. The reactionwas stopped. The reaction mixture was filtered through a pad of Celiteand the black cake was washed with methanol (3×15 ml). The filtrate wasconcentrated and the residue was used in next step directly withoutfurther purification.

Step 5

To a dry 100-ml flask with magnetic stir bar was added the aminesynthesized in step 4 (13.4 mmol), Cbz-L-tyrosine (4.65 g, 14.74 mmol,1.1 eq.), HOBt (2.0 g, 14.74 mmol, 1.1 eq.), anhydrous DMF (40 ml). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(2.83 g, 14.74 mmol, 1.1 eq.). The resulting reaction mixture wasstirred at RT for 16 h. The reaction was monitored by LCMS. The reactionmixture was diluted with EtOAc (500 ml)/water (150 ml). The organicphase was separated and the aqueous phase was extracted by EtOAc (2×100ml). The combined organic layers were washed by water (200 ml), 10%aqueous NaHSO₄ (150 ml), water (150 ml), saturated NaHCO₃ (150 ml), andbrine (2×100 ml), and then dried over Na₂SO₄. After concentration, thecrude was used directly in the next step.

Step 6

An electrochemical cell was assembled using a glass cylinder (6 cmdiameter×11 cm height) and a custom rack (polypropylene and nylon) whichsupported 9 vertical graphite rods (6.15 mm diameter×12 cm length). Therods were arranged in a pattern of a ring with 6 anodes and 3 cathodes.Electrodes were immersed to a depth of 6.5 cm. The phenolic materialsynthesized in Step 5 above (2.00 g, 3.11 mmol), Et₄NBF₄ (2.00 g, 9.2mmol, 3 eq.), K₂CO₃(0.409 g, 2.96 mmol, 0.95 eq.) and ID water (4 ml)were added in DMF (200 ml). The solution was stirred vigorously in astir plate (approx. 600 rpm). The electrochemical reaction was carriedout at a potential of 1.5-1.6 volts. After 3 days, most of the originalSM was consumed as determined by HPLC integration at 220 nM. Theelectrochemistry reaction was repeated for 4 times to consume allphenolic material synthesized in step 5. The combined reaction mixtureswere concentrated on a rotary evaporator (bath temp. ≦35° C.) and driedfurther on a vacuum manifold. The residue was diluted with EtOAc (500ml) followed by filtration through a fritted funnel. The filtrate waswashed by water (2×200 ml) brine (200 ml). The aqueous layers wereextracted in succession with EtOAc (2×50 ml). The combined organiclayers were dried (Na₂SO₄) and concentrated. This material was purifiedby flash column chromatography with 15% MeCN in CH₂Cl₂. This yielded 553mg of desired product with 6.4% yield in three steps.

Step 7

The compound synthesized in Step 6 (553 mg, 0.863 mmol) was dissolved inmethanol (17 ml) and the solution was cooled in an ice bath. A solutionof LiOH (207 mg, 8.63 mmol, 10 eq.) in water (2.7 ml) was added over 5min. The ice bath was removed and the mixture was stirred at RT for 18h. The mixture was cooled in an ice bath and water (20 ml) was addedfollowed by 1 N aqueous HCl (8.8 ml), keeping the reaction temperaturebelow 10° C. The mixture was partitioned between water (15 ml) and EtOAc(100 ml), and the organic layer was washed with saturated aqueous NaCl.The aqueous layers were extracted in succession with EtOAc (30 ml). Thecombined organic layers were dried (Na₂SO₄), decanted, and evaporated togive the acid product as fine white crystals.

Step 8

To a dry 50-ml flask with magnetic stir bar was added the carboxylicacid synthesized in step 7 above (0.863 mmol), L-serine methyl esterhydrochloride (161 mg, 1.036 mmol, 1.2 eq.), HOBt (140 mg, 1.036 mmol,1.2 eq.), anhydrous DMF (15 ml) and N,N-diisopropylethylamine (0.346 ml,1.99 mmol, 2.3 eq.). The reaction mixture was cooled to 0° C. followedby addition of EDC HCl (199 mg, 1.036 mmol, 1.3 eq.). The resultingreaction mixture was stirred at RT for 16 h. The reaction was monitoredby LCMS. Most of solvents were evaporated under reduced pressure. Theresidue was diluted with EtOAc (100 ml)/water (30 ml). The organic phasewas separated and the aqueous phase was extracted by EtOAc (2×20 ml).The combined organic layers were washed by water (40 ml), 10% aqueousNaHSO₄ (40 ml), water (40 ml), saturated NaHCO₃ (40 ml), and brine (2×40ml), and then dried over Na₂SO₄. After concentration, the crude was useddirectly in the next step.

Step 9

To a dry flask were added the crude product from Step 8 above (0.863mmol) and anhydrous CH₂Cl₂ (15 ml). The reaction solution became cloudyas it was cooled to −20° C. in a dry ice/acetone/water bath. A freshlymade stock solution of Bis(2-methoxyethyl)aminosulfur trifluoride (0.239ml, 1.29 mmol, 1.5 eq.) in CH₂Cl₂ (2 ml) was added dropwise. Theresulting reaction mixture was stirred at −20° C. for 1 h, and warmed toroom temperature. The reaction mixture was quenched by addition ofsaturated aqueous NaHCO₃ (10 ml), diluted with EtOAc (50 ml), washedwith water (2×15 ml) as well as brine (20 ml), and dried over Na₂SO₄.After concentration the residue was used in next step.

Step 10

To a dry flask containing the crude product from step 9 above (0.866mmol) were added anhydrous CH₂Cl₂ (15 ml). The mixture was cooled to 0°C. Then CBrCl₃ (0.128 ml, 1.29 mmol, 1.5 eq.) and DBU (0.193 ml, 1.29mmol, 1.5 eq.) were added respectively. The resulting mixture wasallowed to warm to room temperature and was stirred for 1 h. Thereaction was monitored by LCMS. The reaction mixture was diluted withEtOAc (50 ml), washed by 10% NaHSO₄ (15 ml), water (2×15 ml), saturatedaqueous NaHCO₃ (15 ml), water (15 ml) and brine (15 ml), dried overNa₂SO₄. After concentration the residue was used in next step.

Step 11

To a 50-ml flask containing material synthesized in Step 10 above (0.863mmol) were added methanol (12 ml), t-butylamine (0.137 ml, 1.3 mmol, 1.5eq.) and Pd/C (10%) (91 mg, 0.0863 mmol, 0.1 eq.) under N₂. H₂ balloonwas added and the flask was purged with H₂ for 4 times. Then H₂ balloonwas opened to the reaction system. After 4 h stirring almost no startingmaterial remained. The reaction was stopped. The reaction mixture wasfiltered through a pad of Celite and the black cake was washed withmethanol (3×10 ml). The filtrate was concentrated and the residue wasused in next step directly without further purification.

Step 12

To a dry 25-ml flask containing the amine synthesized in Step 11 above(0.863 mmol) were added (S)-(+)-2-hydroxy-3-methylbutanoic acid (122 mg,1.036 mmol, 1.2 eq.), HOBt (140 mg, 1.036 mmol, 1.2 eq.), anhydrous DMF(10 ml) and N,N-diisopropylethylamine (0.225 ml, 1.29 mmol, 1.5 eq.).The reaction mixture was cooled to 0° C. followed by addition of EDC HCl(199 mg, 1.036 mmol, 1.2 eq.). The resulting reaction mixture wasstirred at room temperature for 16 h. The reaction was monitored byLCMS. The reaction mixture was diluted with EtOAc (100 ml)/water (30ml). The organic phase was separated and the aqueous phase was extractedby EtOAc (2×20 ml). The combined organic layers were washed by water (30ml), 10% aqueous NaHSO₄ (30 ml), water (30 ml), saturated NaHCO₃ (30ml), and brine (2×30 ml), and then dried over Na₂SO₄. Afterconcentration, the crude was used directly in the next step.

Step 13

To a dry flask were added crude material synthesized in Step 12 (0.863mmol), THF (10 ml) and 2-propanol (30 ml). This solution was cooled to0° C. followed by addition of solid lithium borohydride (282 mg, 12.95mmol, 15 eq.). The resulting mixture was allowed to warm to roomtemperature and stirred for 22 h. The reaction was monitored with LCMS.Almost no starting material remained. The reaction mixture was cooled to0° C. 2-Propanol (24 ml) and water (40 ml) were added followed byaddition of NH₄Cl (4.6 g, 86.3 mmol, 100 eq.). The reaction mixture wasstirred for 1 h and diluted with EtOAc (250 ml)/water (50 ml). Theorganic phase was separated and the aqueous phase was extracted by EtOAc(2×50 ml). The combined organic layers were washed by water (3×100 ml),10% NaHSO₄ (2×100 ml), water (2×100 ml), saturated NaHCO₃ (100 ml), andbrine (2×100 ml), and then dried over Na₂SO₄. After concentration theresidue was purified by flash column chromatography (70% EtOAc/DCM) toafford desired product as an off-white solid (124 mg, 0.192 mmol, 22%for seven steps).

Synthesis of Compound 86

Step 1

To a dry 250-ml flask were added 5-fluoro-DL-tryptophane (5.0 g, 22.5mmol), and anhydrous methanol (120 ml). The suspension was cooled to 0°C. followed by addition of chlorotrimethyl silane (12.8 ml, 101.3 mmol,4.5 eq.) in such a rate to keep the reaction temperature below 6° C. Theresulting reaction mixture was stirred at room temperature for 20 h. Thereaction was monitored by TLC. Most volatile substances were evaporatedunder reduced pressure. The crude was used in next step.

Step 2

To a dry 250-ml flask with magnetic stir bar was added the amine saltsynthesized in step 1 above (22.5 mmol.), Cbz-L-valine (6.22 g, 24.75mmol, 1.1 eq.), HOBt (3.34 g, 24.75 mmol, 1.1 eq.), anhydrous DMF (80ml) and N,N-diisopropylethylamine (11.8 ml, 67.5 mmol, 3.0 eq.). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(4.74 g, 24.75 mmol, 1.1 eq.). The resulting reaction mixture wasstirred at RT for 16 h. The reaction was monitored by LCMS. Most ofsolvents were evaporated under reduced pressure. Then the residue wasdiluted with EtOAc (600 ml)/water (200 ml). The organic phase wasseparated and the aqueous phase was extracted by EtOAc (2×50 ml). Thecombined organic layers were washed by water (100 ml), 10% aqueousNaHSO₄ (100 ml), water (100 ml), saturated NaHCO₃ (100 ml), and brine(2×100 ml), and then dried over Na₂SO₄. After concentration, the crudewas used directly in the next step.

Step 3

A solution of DDQ (12.8 g, 56.25 mmol, 2.5 eq.) in THF (500 ml) wasadded to the refluxing solution of the compound synthesized in Step 2above (22.5 mmol) in THF (250 ml) and the dark solution was kept inreflux in an oil bath at 85° C. for 1 h. After cooling, the solvent wasremoved on a rotary evaporator. The residue was dissolved in ethylacetate (600 ml), and NaHCO₃ (13 g) was added. The mixture was stirredfor 1 h followed by filtration through a fritted funnel. The filtratewas washed by water (200 ml), aqueous saturated NaHCO₃ (2×200 ml), water(2×200 ml), brine (100 ml) and dried over Na₂SO₄. After concentration,the mixture was purified by flash column chromatography (5% EtOAc inCH₂Cl₂). This yielded 4.63 g (44.2% yield) of product.

Step 4

To a 250-ml flask containing material synthesized in Step 3 above (4.63g, 9.94 mmol) was added methanol (50 ml) and Pd/C (10%) (530 mg, 0.497mmol, 0.05 eq.) under N₂. H₂ balloon was added and the flask was purgedwith H₂ for 4 times. Then H₂ balloon was opened to the reaction system.After 1 h stirring almost no starting material remained. The reactionwas stopped. The reaction mixture was filtered through a pad of Celiteand the black cake was washed with methanol (3×15 ml). The filtrate wasconcentrated and the residue was used in next step directly withoutfurther purification.

Step 5

To a dry 100-ml flask with magnetic stir bar was added the aminesynthesized in step 4 (9.94 mmol), Cbz-L-tyrosine (3.45 g, 10.93 mmol,1.1 eq.), HOBt (1.48 g, 10.93 mmol, 1.1 eq.), anhydrous DMF (30 ml). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(2.10 g, 10.93 mmol, 1.1 eq.). The resulting reaction mixture wasstirred at RT for 16 h. The reaction was monitored by LCMS. The reactionmixture was diluted with EtOAc (400 ml)/water (150 ml). The organicphase was separated and the aqueous phase was extracted by EtOAc (2×100ml). The combined organic layers were washed by water (200 ml), 10%aqueous NaHSO₄ (150 ml), water (150 ml), saturated NaHCO₃ (150 ml), andbrine (2×100 ml), and then dried over Na₂SO₄. After concentration, thecrude (6.58 g) was used directly in the next step.

Step 6

An electrochemical cell was assembled using a glass cylinder (6 cmdiameter×11 cm height) and a custom rack (polypropylene and nylon) whichsupported 9 vertical graphite rods (6.15 mm diameter×12 cm length). Therods were arranged in a pattern of a ring with 6 anodes and 3 cathodes.Electrodes were immersed to a depth of 6.5 cm. The phenolic materialsynthesized in Step 5 above (2.00 g, 3.18 mmol), Et₄NBF₄ (2.00 g, 9.2mmol, 3 eq.), K₂CO₃(0.44 g, 3.18 mmol, 1.0 eq.) and ID water (4 ml) wereadded in DMF (200 ml). The solution was stirred vigorously in a stirplate (approx. 600 rpm). The electrochemical reaction was carried out ata potential of 1.5-1.6 volts. After 3 days, most of the original SM wasconsumed as determined by HPLC integration at 220 nM. Theelectrochemistry reaction was repeated for 4 times to consume allphenolic material synthesized in step 5. The combined reaction mixtureswere concentrated on a rotary evaporator (bath temp. ≦35° C.) and driedfurther on a vacuum manifold. The residue was diluted with EtOAc (500ml) followed by filtration through a fritted funnel. The filtrate waswashed by water (2×200 ml), brine (200 ml). The aqueous layers wereextracted in succession with EtOAc (2×50 ml). The combined organiclayers were dried (Na₂SO₄) and concentrated. This material was purifiedby flash column chromatography with 15% MeCN in CH₂Cl₂. This yielded 900mg of desired product with 14% yield in three steps.

Step 7

The compound synthesized in Step 6 (900 mg, 1.43 mmol) was dissolved inmethanol (28 ml) and the solution was cooled in an ice bath. A solutionof LiOH (344 mg, 14.3 mmol, 10 eq.) in water (4.5 ml) was added over 5min. The ice bath was removed and the mixture was stirred at RT for 18h. The mixture was cooled in an ice bath and water (40 ml) was addedfollowed by 1 N aqueous HCl (14.5 ml), keeping the reaction temperaturebelow 10° C. The mixture was partitioned between water (25 ml) and EtOAc(200 ml), and the organic layer was washed with saturated aqueous NaCl.The aqueous layers were extracted in succession with EtOAc (50 ml). Thecombined organic layers were dried (Na₂SO₄), decanted, and evaporated togive the acid product as fine white crystals.

Step 8

To a dry 50-ml flask with magnetic stir bar was added the carboxylicacid synthesized in step 7 above (1.43 mmol), L-serine methyl esterhydrochloride (268 mg, 1.72 mmol, 1.2 eq.), HOBt (232 mg, 1.72 mmol, 1.2eq.), anhydrous DMF (15 ml) and N,N-diisopropylethylamine (0.624 ml,3.58 mmol, 2.5 eq.). The reaction mixture was cooled to 0° C. followedby addition of EDC HCl (330 mg, 1.72 mmol, 1.2 eq.). The resultingreaction mixture was stirred at RT for 16 h. The reaction was monitoredby LCMS. Most of solvents were evaporated under reduced pressure. Theresidue was diluted with EtOAc (150 ml)/water (50 ml). The organic phasewas separated and the aqueous phase was extracted by EtOAc (2×30 ml).The combined organic layers were washed by water (60 ml), 10% aqueousNaHSO₄ (60 ml), water (60 ml), saturated NaHCO₃ (60 ml), and brine (2×60ml), and then dried over Na₂SO₄. After concentration, the crude was useddirectly in the next step.

Step 9

To a dry flask were added the crude product from Step 8 above (1.43mmol) and anhydrous CH₂Cl₂ (25 ml). The reaction solution became cloudyas it was cooled to −20° C. in a dry ice/acetone/water bath. A freshlymade stock solution of Bis(2-methoxyethyl)aminosulfur trifluoride (0.395ml, 2.15 mmol, 1.5 eq.) in CH₂Cl₂ (4 ml) was added dropwise. Theresulting reaction mixture was stirred at −20° C. for 1 h, and warmed toroom temperature. The reaction mixture was quenched by addition ofsaturated aqueous NaHCO₃ (15 ml), diluted with EtOAc (100 ml), washedwith water (2×20 ml) as well as brine (30 ml), and dried over Na₂SO₄.After concentration the residue was used in next step.

Step 10

To a dry flask containing the crude product from step 9 above (1.43mmol) were added anhydrous CH₂Cl₂ (25 ml). The mixture was cooled to 0°C. Then CBrCl₃ (0.211 ml, 2.15 mmol, 1.5 eq.) and DBU (0.321 ml, 2.15mmol, 1.5 eq.) were added respectively. The resulting mixture wasallowed to warm to room temperature and was stirred for 1 h. Thereaction was monitored by LCMS. The reaction mixture was diluted withEtOAc (100 ml), washed by 10% NaHSO₄ (30 ml), water (2×30 ml), saturatedaqueous NaHCO₃ (15 ml), water (30 ml) and brine (30 ml), dried overNa₂SO₄. After concentration the residue was used in next step.

Step 11

To a flask were added the product from step 10 (0.735 mmol), methanol(30 ml), aqueous ammonia solution (28%, 15 ml). The resulting reactionmixture was stirred at room temperature for 24 hrs. The reaction wasmonitored by TLC. Most of methanol was evaporated under reducedpressure. The residue was extracted by ethyl acetate (3×30 ml), washedwith 2% NaHSO₄ (30 ml), water (30 ml), 5% NaHCO₃ (30 ml), brine (30 ml).The organic phase was dried over Na₂SO₄. After concentration, the crudewas used in next step.

Step 12

To a 100-ml flask containing material synthesized in Step 11 above(0.735 mmol) were added methanol (20 ml), t-butylamine (0.116 ml, 1.1mmol, 1.5 eq.) and Pd/C (10%) (78 mg, 0.052 mmol, 0.1 eq.) under N₂. H₂balloon was added and the flask was purged with H₂ for 4 times. Then H₂balloon was opened to the reaction system. After 4 h stirring almost nostarting material remained. The reaction was stopped. The reactionmixture was filtered through a pad of Celite and the black cake waswashed with methanol (3×15 ml). The filtrate was concentrated and theresidue was used in next step directly without further purification.

Step 13

To a dry 25-ml flask containing the amine synthesized in Step 12 above(0.735 mmol) were added (S)-(+)-2-hydroxy-3-methylbutanoic acid (104 mg,0.882 mmol, 1.2 eq.), HOBt (119 mg, 0.882 mmol, 1.2 eq.), anhydrous DMF(10 ml) and N,N-diisopropylethylamine (0.192 ml, 1.1 mmol, 1.5 eq.). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(169 mg, 0.882 mmol, 1.2 eq.). The resulting reaction mixture wasstirred at room temperature for 16 h. The reaction was monitored byLCMS. The reaction mixture was diluted with EtOAc (100 ml)/water (30ml). The organic phase was separated and the aqueous phase was extractedby EtOAc (2×30 ml). The combined organic layers were washed by water (50ml), 10% aqueous NaHSO₄ (50 ml), water (30 ml), saturated NaHCO₃ (50ml), and brine (2×50 ml), and then dried over Na₂SO₄. Afterconcentration, the crude was used directly in the next step.

Step 14

To a dry flask were added crude material synthesized in Step 13 (0.735mmol), dioxane (10 ml) and CH₂Cl₂ (10 ml) and pyridine (1.2 ml, 14.7mmol). This solution was cooled to −17° C. followed by addition oftrifluoroacetic anhydride (1.5 ml, 11 mmol) at −10 to −17° C. Afteraddition, the resulting mixture was stirred at −15° C. for 1 h. Thenaqueous NH₃ solution (28% 10 ml) was added dropwise at −15° C. followedby warming to room temperature and stirred for 1 h. The reaction wasmonitored with LCMS. Most of solvent was moved under reduced pressure.The residue was extracted by ethyl acetate (3×20 ml), washed with water(2×20 ml), 5% NaHSO₄ (20 ml), water (20 ml), sat. NaHCO₃ (20 ml), water(20 ml) and brine (20 ml). The organic phase was dried over Na2SO4.After concentration the residue was purified by flash columnchromatography (30% MeCN/DCM to 40% MeCN/DCM) to afford desired productas an off-white solid (115 mg, 0.184 mmol, 25% for eight steps).

Synthesis of Compound 87

Step 1

To a dry 250-ml flask were added 5-fluoro-DL-tryptophane (6.0 g, 27.0mmol), and anhydrous methanol (120 ml). The suspension was cooled to 0°C. followed by addition of chlorotrimethyl silane (15.4 ml, 121.5 mmol,4.5 eq.) in such a rate to keep the reaction temperature below 6° C. Theresulting reaction mixture was stirred at room temperature for 20 h. Thereaction was monitored by TLC. Most volatile substances were evaporatedunder reduced pressure. The crude was used in next step.

Step 2

To a dry 250-ml flask with magnetic stir bar was added the amine saltsynthesized in step 1 above (27 mmol.), Cbz-L-α-t-butylglycine DCHA salt(13.26 g, 29.7 mmol, 1.1 eq.), HOBt (4.01 g, 29.7 mmol, 1.1 eq.),anhydrous DMF (100 ml) and N,N-diisopropylethylamine (14.1 ml, 81 mmol,3.0 eq.). The reaction mixture was cooled to 0° C. followed by additionof EDC HCl (5.69 g, 29.7 mmol, 1.1 eq.). The resulting reaction mixturewas stirred at RT for 16 h. The reaction was monitored by LCMS. Most ofsolvents were evaporated under reduced pressure. Then the residue wasdiluted with EtOAc (700 ml)/water (200 ml). The organic phase wasseparated and the aqueous phase was extracted by EtOAc (2×50 ml). Thecombined organic layers were washed by water (100 ml), 10% aqueousNaHSO₄ (100 ml), water (100 ml), saturated NaHCO₃ (100 ml), and brine(2×100 ml), and then dried over Na₂SO₄. After concentration, the crudewas used directly in the next step.

Step 3

A solution of DDQ (15.32 g, 67.5 mmol, 2.5 eq.) in THF (100 ml) wasadded to the refluxing solution of the compound synthesized in Step 2above (27 mmol) in THF (300 ml) and the dark solution was kept in refluxin an oil bath at 85° C. for 1 h. After cooling, the solvent was removedon a rotary evaporator. The residue was dissolved in ethyl acetate (700ml), and NaHCO₃ (15 g) was added. The mixture was stirred for 1 hfollowed by filtration through a fritted funnel. The filtrate was washedby water (200 ml), aqueous saturated NaHCO₃ (2×200 ml), water (2×200ml), brine (100 ml) and dried over Na₂SO₄. After concentration, themixture was purified by flash column chromatography (5% EtOAc inCH₂Cl₂). This yielded 6.42 g (50% yield) of product.

Step 4

To a 250-ml flask containing material synthesized in Step 3 above (6.42g, 13.4 mmol) was added methanol (60 ml) and Pd/C (10%) (1.43 g, 1.34mmol, 0.1 eq.) under N₂. H₂ balloon was added and the flask was purgedwith H₂ for 4 times. Then H₂ balloon was opened to the reaction system.After 1 h stirring almost no starting material remained. The reactionwas stopped. The reaction mixture was filtered through a pad of Celiteand the black cake was washed with methanol (3×15 ml). The filtrate wasconcentrated and the residue was used in next step directly withoutfurther purification.

Step 5

To a dry 100-ml flask with magnetic stir bar was added the aminesynthesized in step 4 (13.4 mmol), Cbz-L-tyrosine (4.65 g, 14.74 mmol,1.1 eq.), HOBt (2.0 g, 14.74 mmol, 1.1 eq.), anhydrous DMF (40 ml). Thereaction mixture was cooled to 0° C. followed by addition of EDC HCl(2.83 g, 14.74 mmol, 1.1 eq.). The resulting reaction mixture wasstirred at RT for 16 h. The reaction was monitored by LCMS. The reactionmixture was diluted with EtOAc (500 ml)/water (150 ml). The organicphase was separated and the aqueous phase was extracted by EtOAc (2×100ml). The combined organic layers were washed by water (200 ml), 10%aqueous NaHSO₄ (150 ml), water (150 ml), saturated NaHCO₃ (150 ml), andbrine (2×100 ml), and then dried over Na₂SO₄. After concentration, thecrude was used directly in the next step.

Step 6

An electrochemical cell was assembled using a glass cylinder (6 cmdiameter×11 cm height) and a custom rack (polypropylene and nylon) whichsupported 9 vertical graphite rods (6.15 mm diameter×12 cm length). Therods were arranged in a pattern of a ring with 6 anodes and 3 cathodes.Electrodes were immersed to a depth of 6.5 cm. The phenolic materialsynthesized in Step 5 above (2.00 g, 3.11 mmol), Et₄NBF₄ (2.00 g, 9.2mmol, 3 eq.), K₂CO₃(0.409 g, 2.96 mmol, 0.95 eq.) and ID water (4 ml)were added in DMF (200 ml). The solution was stirred vigorously in astir plate (approx. 600 rpm). The electrochemical reaction was carriedout at a potential of 1.5-1.6 volts. After 3 days, most of the originalSM was consumed as determined by HPLC integration at 220 nM. Theelectrochemistry reaction was repeated for 4 times to consume allphenolic material synthesized in step 5. The combined reaction mixtureswere concentrated on a rotary evaporator (bath temp. ≦35° C.) and driedfurther on a vacuum manifold. The residue was diluted with EtOAc (500ml) followed by filtration through a fritted funnel. The filtrate waswashed by water (2×200 ml) brine (200 ml). The aqueous layers wereextracted in succession with EtOAc (2×50 ml). The combined organiclayers were dried (Na₂SO₄) and concentrated. This material was purifiedby flash column chromatography with 15% MeCN in CH₂Cl₂. This yielded 553mg of desired product with 6.4% yield in three steps.

Step 7

The compound synthesized in Step 6 (553 mg, 0.863 mmol) was dissolved inmethanol (17 ml) and the solution was cooled in an ice bath. A solutionof LiOH (207 mg, 8.63 mmol, 10 eq.) in water (2.7 ml) was added over 5min. The ice bath was removed and the mixture was stirred at RT for 18h. The mixture was cooled in an ice bath and water (20 ml) was addedfollowed by 1 N aqueous HCl (8.8 ml), keeping the reaction temperaturebelow 10° C. The mixture was partitioned between water (15 ml) and EtOAc(100 ml), and the organic layer was washed with saturated aqueous NaCl.The aqueous layers were extracted in succession with EtOAc (30 ml). Thecombined organic layers were dried (Na₂SO₄), decanted, and evaporated togive the acid product as fine white crystals.

Step 8

To a dry 50-ml flask with magnetic stir bar was added the carboxylicacid synthesized in step 7 above (0.863 mmol), L-serine methyl esterhydrochloride (161 mg, 1.036 mmol, 1.2 eq.), HOBt (140 mg, 1.036 mmol,1.2 eq.), anhydrous DMF (15 ml) and N,N-diisopropylethylamine (0.346 ml,1.99 mmol, 2.3 eq.). The reaction mixture was cooled to 0° C. followedby addition of EDC HCl (199 mg, 1.036 mmol, 1.3 eq.). The resultingreaction mixture was stirred at RT for 16 h. The reaction was monitoredby LCMS. Most of solvents were evaporated under reduced pressure. Theresidue was diluted with EtOAc (100 ml)/water (30 ml). The organic phasewas separated and the aqueous phase was extracted by EtOAc (2×20 ml).The combined organic layers were washed by water (40 ml), 10% aqueousNaHSO₄ (40 ml), water (40 ml), saturated NaHCO₃ (40 ml), and brine (2×40ml), and then dried over Na₂SO₄. After concentration, the crude was useddirectly in the next step.

Step 9

To a dry flask were added the crude product from Step 8 above (0.863mmol) and anhydrous CH₂Cl₂ (15 ml). The reaction solution became cloudyas it was cooled to −20° C. in a dry ice/acetone/water bath. A freshlymade stock solution of Bis(2-methoxyethyl)aminosulfur trifluoride (0.239ml, 1.29 mmol, 1.5 eq.) in CH₂Cl₂ (2 ml) was added dropwise. Theresulting reaction mixture was stirred at −20° C. for 1 h, and warmed toroom temperature. The reaction mixture was quenched by addition ofsaturated aqueous NaHCO₃ (10 ml), diluted with EtOAc (50 ml), washedwith water (2×15 ml) as well as brine (20 ml), and dried over Na₂SO₄.After concentration the residue was used in next step.

Step 10

To a dry flask containing the crude product from step 9 above (0.866mmol) were added anhydrous CH₂Cl₂ (15 ml). The mixture was cooled to 0°C. Then CBrCl₃ (0.128 ml, 1.29 mmol, 1.5 eq.) and DBU (0.193 ml, 1.29mmol, 1.5 eq.) were added respectively. The resulting mixture wasallowed to warm to room temperature and was stirred for 1 h. Thereaction was monitored by LCMS. The reaction mixture was diluted withEtOAc (50 ml), washed by 10% NaHSO₄ (15 ml), water (2×15 ml), saturatedaqueous NaHCO₃ (15 ml), water (15 ml) and brine (15 ml), dried overNa₂SO₄. After concentration the residue was used in next step.

Step 11

To a flask were added the product from step 10 (0.52 mmol), methanol (25ml), aqueous ammonia solution (28%, 10 ml). The resulting reactionmixture was stirred at room temperature for 24 hrs. The reaction wasmonitored by TLC. Most of methanol was evaporated under reducedpressure. The residue was extracted by ethyl acetate (3×30 ml), washedwith 2% NaHSO₄ (30 ml), water (30 ml), 5% NaHCO₃ (30 ml), brine (30 ml).The organic phase was dried over Na₂SO₄. After concentration, the crudewas used in next step.

Step 12

To a 100-ml flask containing material synthesized in Step 11 above (0.52mmol) were added methanol (10 ml), t-butylamine (0.082 ml, 0.78 mmol,1.5 eq.) and Pd/C (10%) (55 mg, 0.052 mmol, 0.1 eq.) under N₂. H₂balloon was added and the flask was purged with H₂ for 4 times. Then H₂balloon was opened to the reaction system. After 4 h stirring almost nostarting material remained. The reaction was stopped. The reactionmixture was filtered through a pad of Celite and the black cake waswashed with methanol (3×15 ml). The filtrate was concentrated and theresidue was used in next step directly without further purification.

Step 13

To a dry 25-ml flask containing the amine synthesized in Step 12 above(0.52 mmol) were added (S)-(+)-2-hydroxy-3-methylbutanoic acid (74 mg,0.624 mmol, 1.2 eq.), HOBt (85 mg, 0.624 mmol, 1.2 eq.), anhydrous DMF(10 ml) and N,N-diisopropylethylamine (0.136 ml, 0.78 mmol, 1.5 eq.).The reaction mixture was cooled to 0° C. followed by addition of EDC HCl(120 mg, 0.624 mmol, 1.2 eq.). The resulting reaction mixture wasstirred at room temperature for 16 h. The reaction was monitored byLCMS. The reaction mixture was diluted with EtOAc (100 ml)/water (30ml). The organic phase was separated and the aqueous phase was extractedby EtOAc (2×30 ml). The combined organic layers were washed by water (50ml), 10% aqueous NaHSO₄ (50 ml), water (30 ml), saturated NaHCO₃ (50ml), and brine (2×50 ml), and then dried over Na₂SO₄. Afterconcentration, the crude was used directly in the next step.

Step 14

To a dry flask were added crude material synthesized in Step 13 (0.52mmol), dioxane (7 ml) and CH₂Cl₂ (7 ml) and pyridine (0.841 ml, 14.7mmol). This solution was cooled to −17° C. followed by addition oftrifluoroacetic anhydride (1.1 ml, 7.8 mmol) at −10 to −17° C. Afteraddition, the resulting mixture was stirred at −15° C. for 1 h. Thenaqueous NH₃ solution (28% 7 ml) was added dropwise at −15° C. followedby warming to room temperature and stirred for 1 h. The reaction wasmonitored with LCMS. Most of solvent was moved under reduced pressure.The residue was extracted by ethyl acetate (3×20 ml), washed with water(2×20 ml), 5% NaHSO₄ (20 ml), water (20 ml), sat. NaHCO₃ (20 ml), water(20 ml) and brine (20 ml). The organic phase was dried over Na₂SO₄.After concentration the residue was purified by flash columnchromatography (20% MeCN/DCM to 30% MeCN/DCM) to afford desired productas an off-white solid (51 mg, 0.080 mmol, 16% for eight steps).

Cell Viability Assay Protocol

Cell viability assays were run using standard protocols known to thoseof skill in art. Cells were plated in 96 well plates at the density of3,000-10,000 cells per well. Twenty four hours later, cells were treatedwith increasing concentration of test compounds (1 nM to 1 μM). Afteranother 48 hour, cell survival was measured using Cell-Titer-Glo®reagent (Promega) following the protocol provided by the manufacture.The IC₅₀ value was determined as the concentration of test compound thatkills 50% of the cell population.

Representative Biological Data

Cell viability data generated according to the protocol described abovewas generated for representative compounds in A2058 and U937 cells. Thecompounds shown in Table 1 were prepared by the methods described hereinfor structurally similar compounds. The reference compound was asynthetic diazonamide analog having the structure:

TABLE 1 Cell viability data in A2058 and U937 cells IC50 (nM) IC50 (nM)in in cell cell viability viability assay in assay in Compound #Structure A2058 U937 I. Oxazole, 4 oxazoyl analogs with esters otherthan methyl ester in position 4. 21

40 20 22

52 19 23

57 21 24

25.99 19.68 25

70.81 36.91 26

3.33 2.88 2. Oxazole, 4 oxazoyl analogs with alcohol or ketone inposition 4 27

112 98 28

143 112 29

119 59 30

60 13 31

602 259 III. Oxazole, 4 oxazoyl analogs with amide, amine, carbamate orsulfonamide in position 4 32

>1000 >1000 33

>1000 >1000 34

149.1 133.5 35

150.8 198 36

194 209.3 37

235.21 177.9 IV. Oxazole, 4 oxazoyl analogs with cyano-group in position4 38

>1000 >1000 39

23 71 40

181.6 209.89 V. Oxazole, 4 oxazoyl analogs with heterocycles in position4 41

65 65 42

49 43 43

22 19 44

>1000 >1000 45

589 318 46

49.04 76.88 47

7.06 14.16 48

6.11 6.34 VI. Oxazole, 4 oxazoyl analogs with substituents replacingisopropyl group 49

>1000 223 50

53 12 51

>1000 >1000 52

257 71 53

>1000 >1000 54

>1000 >1000 55

>1000 >1000 56

968 588 57

8.3 5.3 58

>1000 445 59

221 69 60

>1000 795 61

>1000 761 62

2.54 15.64 63

15.5 1.97 64

43.22 45.92 65

16.82 67.56 66

186.16 214.9 67

35.78 89.7 68

21.07 59.6 69

95.52 225.2 7. Oxazole, 4 oxazoyl analogs with variations in tyrosinemoiety 70

77 15 71

67 9.2 72

>300 213.05 73

<0.1 <0.1 74

>100 >100 75

1.89 6.55 8. Oxazole, 4 oxazoyl analogs with variations in indolinemoiety 76

>1000 614 77

>1000 777 78

113.8 17.39 79

270 209.6 80

13.58 4.07 81

55.08 32.77 82

40.91 3.4 83

183.87 60.6 84

19.51 10.13 85

0.47 0.74 86

0.56 21.78 87

<0.3 1.9 IX. Oxazole, 4 oxazoyl analogs with N-Methyl group in sidechain and/or macrocyclic moiety 88

0.8 0.6 89

3 1 90

10.4 17.12 91

0.59 1.97 92

113.35 69.88 93

35.75 18.57 94

>300 >300 X. Oxazole, 4-aryl (non-oxazole) analogs 95

142 65 96

>1000 >1000 97

224.96 59.51 98

284.3 231.3 11. Oxazole, 4 oxazoyl analogs 99

>1000 >1000 100

465 220 101

684 500 102

492 225 103

221 69 104

>300 >300 105

>300 >300

Xenograft Tumor Models

The compounds were tested in HCC461 human lung carcinoma xenograft andMiapaca pancreatic cancer xenograft tumor models in 5- to 6-week-oldHarlan Athymic Nude-Foxnlnu mice.

Protocol:

Preparation of Tumor Cells

Tumor cells were cultured in complete RPMI medium and excluded anycontamination. When cells are 70-80% confluent, medium was removed andcells were washed with serum free media, trypsinized, harvested andwashed with serum free media for three times by centrifuge. After finalwashing, cells were counted and mixed with matrigel at 1:1 ration involume. Cells were suspended in a volume that 200 μl contains requirednumber of cells per injection.

Preparation of the Injection

Clean and sterilize the inoculation area of the mice with iodinesolutions and ethanol. Take cells with1-cc syringe. Inject tumor cells(1×10⁷) subcutaneously (s.c.) into the lower flank of the mice. Whentumors reached 200-300 mm³ in size, mice were randomized into treatmentgroups of five mice per group. Mice were weighed and tumors measuredusing vernier calipers two times per week. Tumor volume in mm³ iscalculated by the formula: Volume (mm³)=(length×width²)/2.

Treatment

The compounds were dissolved in cremophor/ethanol (1:1) at 20 mg/mL asthe stock solution and then diluted in saline to 2.5 mg/mL. Thecompounds and the vehicle (6.25% cremophor/6.25% ethanol in saline) wereadministered intravenously in a total volume of 0.2 mL three times aweek for total six treatments.

In the HCC461 lung cancer xenograft model, animals were injected on days7, 11, 14 and 18 post tumor-cell injection.

In the Miapaca pancreatic cancer xenograft model, animals were injectedon days 6, 13, and 20 post tumor-cell injection.

Results

The activities of exemplary compounds and dosages are shown in FIGS. 1and 2.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the invention. Although the invention has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, and yet these modifications and improvements are within thescope and spirit of the invention.

The invention claimed is:
 1. A compound of the following table, or apharmaceutically acceptable salt thereof: I. Oxazole, 4 oxazoyl analogswith esters other than methyl ester in position 4 21

22

23

24

25

26

II. Oxazole, 4 oxazoyl analogs with alcohol or ketone in position 4 27

28

29

30

31

III. Oxazole, 4 oxazoyl analogs with amide, amine, carbamate orsulfonamide in position 4 32

33

34

35

36

37

IV. Oxazole, 4 oxazoyl analogs with cyano-group in position 4 38

39

40

V. Oxazole, 4 oxazoyl analogs with heterocycles in position 4 41

42

43

44

45

46

47

48

VI. Oxazole, 4 oxazoyl analogs with substituents replacing isopropylgroup 49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

VII. Oxazole, 4 oxazoyl analogs with variations in tyrosine moiety 70

71

72

73

74

75

VIII. Oxazole, 4 oxazoyl analogs with variations in indoline moiety 76

77

78

79

80

81

82

83

84

85

86

87

IX. Oxazole, 4 oxazoyl analogs with N-Methyl group in side chain and/ormacrocyclic moiety 88

89

90

91

92

93

94

X. Oxazole, 4-aryl (non-oxazole) analogs 95

96

97

98

XI. Oxazole, 4 oxazoyl analogs 99

100

101

102

103

104

105


2. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: I. Oxazole, 4 oxazoyl analogswith esters other than methyl ester in position
 4. 21

22

23

24

25

26


3. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: II. Oxazole, 4 oxazoyl analogswith alcohol or ketone in position 4 27

28

29

30

31


4. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: 32

33

34

35

36

37


5. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: IV. Oxazole, 4 oxazoyl analogswith cyano-group in position 4 38

39

40


6. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: V. Oxazole, 4 oxazoyl analogswith heterocycles in position 4 41

42

43

44

45

46

47

48


7. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: VI. Oxazole, 4 oxazoyl analogswith substituents replacing isopropyl group 49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69


8. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: VII. Oxazole, 4 oxazoylanalogs with variations in tyrosine moiety 70

71

72

73

74

75


9. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: VIII. Oxazole, 4 oxazoylanalogs with variations in indoline moiety 76

77

78

79

80

81

82

83

84

85

86

87


10. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: IX. Oxazole, 4 oxazoyl analogswith N-Methyl group in side chain and/or macrocyclic moiety 88

89

90

91

92

93

94


11. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: X. Oxazole, 4-aryl(non-oxazole) analogs 95

96

97

98


12. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof: XI. Oxazole, 4 oxazoyl analogs99

100

101

102

103

104

105


13. A compound of claim 1 and of the following table, or apharmaceutically acceptable salt thereof:


14. A compound of claim 1 having the following formula, or apharmaceutically acceptable salt thereof:


15. A compound of claim 1 having the following formula, or apharmaceutically acceptable salt thereof:


16. A compound of claim 1 having the following formula, or apharmaceutically acceptable salt thereof:


17. A compound of claim 1 having the following formula, or apharmaceutically acceptable salt thereof:


18. A compound of claim 1 having the following formula, or apharmaceutically acceptable salt thereof:


19. A pharmaceutical composition comprising (a) the compound of claim 1in unit dosage form with at least one pharmaceutically acceptableexcipient, or (b) the compound of claim 1 and a differentchemotherapeutic drug.
 20. A method of treating colon cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of claim 1.